The Biosynthesis of β-Glucans in Cotton (Gossypium hirsutum L.) Fibres of Ovules Cultured in vitro

J.PlantPhysiol. Vol. 134.pp. 485-491 (1989)

The Biosynthesis of /3-Glucans in Cotton (Gossypium hirsutum L.) Fibres of Ovules Cultured in vitro Y.

FRANCEY, J.

P. JAQUET, S.

CAIROLl,

A. J.

BUCHALA\

and H.

MEIER

Institut de Biologie vegcftale et de Phytochimie, Universite de Fribourg, CH-1700 Fribourg, Switzerland * To whom correspondence should be addressed. Received November 24, 1988 . Accepted January 18, 1989

Summary When [14C]glucose is fed to the culture medium of cotton ovules cultured in vitro, both iJ-glucans cellulose and callose are synthesised. At the secondary cell wall stage of fibre development, the radioactivity normally incorporated into cellu10se is about twice that found in the callose of the fibres. Pulse-chase experiments showed that callose underwent turnover. Simultaneous application of the herbicide, 2,6-dichlorobenzonitrile, with [14C]glucose significantly inhibited the synthesis of cellulose, but had little effect on the synthesis of callose. Radioactivity in the low-molecular-weight sugar pools (simple sugars, sugar phosphates and sugar nucleotides) of the fibres was higher after treatment with the herbicide, but the latter does not appear to inhibit cytoplasmic enzymes involved in sugar metabolism, suggesting that the inhibitor acts at the plasmalemma itself.

Key words: Gossypium, callose, cellulose, 2,6-dichlorobenzonitrile, iJ-glucan synthesis, turnover. Abbreviations: DCB

=

2,6-dichlorobenzonitrile; DMSO

Introduction It is generally accepted that there are two UDP-glucose: iJglucan synthase activities at the plasma membrane of plant cells (Delmer 1987), but their relationship is all but clear. Freeze-thawing of membranes from celery petioles results in the loss of ability to synthesise (l-+4)-iJ-glucan but not (1-+3)-iJ-glucan Gacob and Northcote 1985). However, these workers did not show that the original (1-+4)-iJ-glucan synthase activity was localised at the plasma membrane. In some cases, formation of (1-+3)-iJ-glucan is clearly the result of cell damage or perturbation (cf Kauss 1987), but in others, e. g. growing pollen tubes (Rae et aI., 1985) and growing cotton fibres (Buchala and Meier 1985), the callose is most likely of non-traumatic origin. Earlier studies carried out on cotton fibres, where the conditions were adjusted so as to be as close as possible to the situation in vivo, clearly showed that the (1-+3 )-iJ-glucan underwent turnover and it was suggested that callose could be an intermediate in the synthesis of cellulose (Meier et aI., 1981). On the other hand, Maltby et aI., (1979) could find no evidence for the turnover of callose in © 1989 by Gustav Fischer Verlag, StUttgart

=

dimethyl-sulphoxide.

cotton fibres grown in vitro in ovule cultures. Such differences prompted a re-examination of the incorporation of [l4C]glucose into the iJ-glucans of ovule-grown fibres. It could be shown that callose does indeed undergo turnover. We have also studied the effect of the herbicide, 2,6-dichlorobenzonitrile (DCB), and some other potential inhibitors on the synthesis of the fibre cell wall iJ-glucans.

Materials and Methods Plant material Cotton plants (Gossypium hirsutum L.) var. Stoneville No. 406 (glandless) were cultivated in a growth chamber at a temperature of 26°C during the day (12.5 h) and 18 °C at night.

Ovule cultures Unfertilised ovules, harvested two days after anthesis, were cultivated on a sterile liquid medium (Beasley and Ting, 1973) supplemented with 511M indolylacetic acid and 511M gibberellic acid at

486

Y. FRANCEY, J. P. JAQUET, S. CAIROU, A. J. BUCHALA, and H. MEIER

34°C in the darkness. Each culture dish normally contained, in four separate compartments, the ovules (about 30) from the four loculi of one ovary.

Feeding of radioactive sugars At the selected age, the ovules from one culture dish were rinsed 3 times with fresh culture medium and then placed in the appropriate incubation medium (20 ml; pH 5.0) where the final sugar concentration was 60 mM. In most experiments the sugar being fed was normally applied at a final concentration of 10 - 20 mM, and the solution was made up to 60 mM with pentaerythritol (see the results section for the details of individual experiments). Labelled sugars, [U}4C]glucose, [U}4C]fructose or [U.14C]sucrose, were added to the culture medium to give 92.5 - 370 kBq/ ml. When the effect of inhibitors was studied, the ovules were preincubated for 1 h in the appropriate incubation medium containing the inhibitor, but no labelled substrate. In pulse-chase experiments, after the pulse the ovules were washed with 3 changes of the incubation medium containing unlabelled sugars, transferred to the chase medium (20 ml) and finally incubated as described above.

Analysis of the incorporation into low-molecular-weight products At the end of the incubation period the aerial fibres were cut off with scissors and immediately plunged into 80 % boiling methanol (or 80% ethanol when sugar nucleotides were to be analysed). After 5 minutes the fibres were recovered by filtration through glass fibre paper (What man GF/ A) and finely cut with a razor blade. The extraction was repeated 5 times and the pooled filtrates and fibres recovered. The filtrate was evaporated to dryness, dissolved in water (4 ml) and the solution extracted twice with 0.5 ml diethylether. Aliquots of the aqueous solution were analysed chromatographically (see below) for neutral sugars, sugar phosphates and sugar nucleotides, where appropriate. Quantitative analyses of glucose, fructose, sucrose after inversion, glucose 6-phosphate, glucose I-phosphate and fructose 6-phosphate were carried out by the hexokinase method (Klotzsch and Bergmeyer 1965). The relative amount of UDP-sugars was estimated by measuring the absorption at 254 nm after separation by HPLC (see below).

Analysis of the high-molecular·weight products The fibres obtained above were suspended in water and freezedried. The dry weight was determined after desiccation over P20 S• Aliquots (5 mg) of the fibres were hydrolysed by the 72 %/4% H 2S04 procedure (Saemen et aI., 1963) and the sugars in hydrolysates examined by paper chromatography. Glucose in hydrolysates was estimated by the Glucose-Perid method (Boehringer, Mannheim, FRG). The callose content of the cotton fibres was determined by extraction with hot dimethyl sulphoxide (DMSO) in the autoclave as described by Pillonel et al. (1980). The residue was considered to consist essentially of cellulose. Quantitative analysis was by total acid hydrolysis followed by determination of the glucose released (see above). The material solubilised in hot DMSO (callose) and the residue obtained after treatment with hot DMSO were characterised by enzymic hydrolysis using an exo-(1-3)-IJ-D-glucanase from the Basidiomycete sp. QM 806 and an exo-(1-4)-IJ-D-glucan cellobiohydrolase from Sporotrichum thermophile as described by Pillonel and Meier (1985). The material solubilised in hot DMSO was also characterised by methylation analysis. The material was permethylated as described

by Jansson et al. (1976) with a radiochemical yield of >80%, and the products obtained upon depolymerisation were examined as their alditol acetate derivatives by capillary gas liquid chromatography (Packard 827) on OV-225 (WCOT 25 m x 0.2 mm) and by proportional counting radio gas chromatography (Packard 894).

Enzyme assays Cotton fibres from fruits harvested 24 days post-anthesis were homogenised in liquid nitrogen and the homogenate was treated with 100 mM Tris buffer (pH 7.5) containing 4 mM dithiothreitol at 4°C in a glass/Teflon homogeniser. The suspension was centrifuged (15 min, 40,000 x g) and the supernatant used for enzymic assays in the presence and absence of 350 ~ DCB. The following activities were assayed as described in the literature: uridine diphosphate glucose pyrophosphorylase (Heiniger and Franz, 1980), hexokinase, phosphoglucomutase, glucose 6-phosphate dehydrogenase (Schnarrenberger et al., 1983), IJ-glucosidase and exo-(1-3)-IJglucanase (Bucheli et al., 1985). (1-3)-IJ-Glucan synthase was assayed in a microsomal membrane pellet as described by Heiniger and Delmer (1977).

Chromatography Monosaccharides were separated and identified by paper chromatography on Schleicher and Schull (Dassel, FRG) No. 2043 b paper using ethyl acetate: pyridine: water (8: 2: 1, by vol.) and by thin layer chromatography on Kieselgel 60 (Merck, Darmstadt, FRG) with acetone: water (88: 12 v/v). Methyl sugars were separated by thin layer chromatography on Kieselgel 60 with benzene: ethanol:water: acetic acid (200: 47: 15: 1, by vol.). Sugars on chromatograms were visualised with alkaline silver nitrate (Trevelyan et al., 1950) or a-naphthol/cone. H 2S0 4 (Stahl 1969) and radioactive sugars were detected using a Linear Analyser LB282 (Berthold, Wildbad, FRG). HPLC (LKB, Bromma, Sweden) of sugar nucleotides and sugar phosphates was carried out on columns (25 em x 4 mm) of Partisil10 SAX (Whatman, Maidstone, UK) using a phosphate buffer system (Carpita and Delmer 1981). Detection was at 254nm and the radioactivity was measured in fractions by liquid scintillation counting.

Results When labelled glucose, fructose or sucrose are added to the culture medium of cotton ovules with fibres at the secondary cell wall stage of development, i. e. at 15 to 20 days post-anthesis, radioactivity is incorporated into the cell wall t1-glucans (callose and cellulose) of the aerial fibres. No other polysaccharides are significantly labelled. In short-term (up to at least 8 h) continuous feeding experiments about 25 % of the radioactivity was incorporated into callose (insoluble in hot 80 % methanol, but soluble in DMSO). At a sugar concentration of 5 mM, glucose is the preferred precursor, the incorporation from labelled fructose and sucrose being 14% and 23 %, that from glucose respectively. At 60 mM, similar percentage values were obtained. The incorporation from [l4C]glucose was examined in detail. The glucose concentration varied between 1 and 90 mM while the osmotic value of the incubation medium was maintained at 120 mM with pentaerythritol. Incorporation of radioactivity into cell wall t1-glucan for a 2 h period (see Fig. 1)

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487

belled glucose (8.4 %) and labelled cellobiose (20.1 %) were released. The residue insoluble in DMSO thus contains labelled (1-+4)-~-glucan. Clearly such a partial characterisation is not sufficient to designate the labelled product as cellulose per se, but the product appears to be indistinguishable from cellulose. The kinetics of labelling of cellulose and callose were examined since it has been shown that callose does undergo turnover in vivo and in certain experimental systems in vitro (Meier et al., 1981). In short-term labelling experiments, incorporation of the radioactivity into both ~-glucans became essentially linear with time after about 20 minutes of incubation and remained more or less linear for up to at least 8 h. (see Fig. 2). The initial lag phases observed for both products precludes any conclusion concerning the eventual role of callose as a direct intermediate in the synthesis of cellulose and may be due to the time necessary to label the cell sugar

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c increases significantly with the glucose concentration up to about 20 mM glucose, and then the incorporation tails off up to about 90 mM indicating saturation of the sugar uptake system or eventually of metabolic glucose pools. The ratio of labelled cellulose to labelled callose was found to be about 2 : 1 at all glucose concentrations. The high-molecular-weight ~-glucans were partially characterized as follows. The material soluble in DMSO was treated with the exo-(1-+3)-~-glucanase from the Basidiomycete sp. QM 806 to give principally glucose, gentiobiose and laminaribiose (6% disaccharides). No radioactivity was released upon treatment with the endo-(1-+3), (1-+4)-~­ glucanase from Bacillus subtilis. The material was methylated Oannson et al. 1976) and the depolymerised permethylated product was examined by thin layer chromatography; 76 % of the radioactivity was found in tri-O-methylglucose, 5 % in 2,3,4,6-tetra-O-methylglucose and 19 % in di-Omethylglucose. The mixture of methyl sugars was reduced with sodium borohydride, acetylated and analysed by capillary gas liquid chromatography. Derivatives of many methyl sugars were detected, but the major components were those expected from a (1-+3)-linked glucan with some branching at 06 of the main chain residues; 2,3,4,6-tetra-0methylglucose, 2,4,6-tri-O-methylglucose and 2,4-di-0methylglucose were present in the ratio (moles %) 3.5: 85.4 : 7.8. In addition, another peak corresponding to 4,6-di-0-methyl- or 2,6-di-O-methylglucose (3.3 %, unresolved) was detected, but its structural significance (branching or undermethylation) is not evident. The material was also examined by radio gas liquid chromatography where the distribution of the radioactivity in the aforementioned peaks was very similar to the mass ratio. Only the peaks corresponding to the products derived from callose were found to be labelled. The material insoluble in hot DMSO was not degraded by the exo-(1-+3)-~-glucanase nor by the endo-(1-+3), (1-+4)-11glucanase, but upon treatment with an exocellobiohydrolase from Sporotrichum thermophile (Canevascini et al., 1983), la-

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Y. FRANCEY, J. P. ]AQUET, S. CAIROLI, A. J. BucHALA, and H. MEIER

488

pools. For longer incubation periods of up to 4 days (see Fig. 3) the incorporation of radioactivity into cellulose increases linearly while the rate of incorporation into callose declines after about 1 day and stabilises after about 2 days. Pulse-chase experiments were also carried out. After a relatively short pulse (1 h) with [14C]glucose only a decline in the rate of incorporation into both of the !3-glucans was observed in the following chase (not shown). After a pulse of 3 h with [l4C]glucose followed by a 2 h chase, turnover, of callose was observed (Fig. 4). Saturation of the metabolic cell sugar pools with radioactivity is probably necessary in order to observe callose turnover. When a pulse of [14C]glucose was applied for 2 days this latter condition was almost certainly satisfied and turnover of callose was clearly observed during the ensuing chase of 3 days (see Fig. 5).

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Experiments with inhibitors of synthesis of !3-glucans were carried out with the aim of elucidating the role of (1-3)-13glucan. 2,6-Dichlorobenzonitrile (DCB) has been reported to specificically inhibit the synthesis of cellulose in cotton fibres (Montezinos and Delmer, 1980; Pillonel and Meier, 1985). We found that a pre-treatment of 1 h with the inhibitor was necessary in order to obtain reproducible results. Preliminary experiments with 14C-Iabelled DCB showed that uptake of DCB from the incubation medium ceased after about 2 h. Concentrations of 10 to 20/.tM DCB in the incubation medium gave rise to about 25 % inhibition of incorporation of radioactivity into cellulose after one to three hours, but had little effect on the incorporation into callose (Table 1 and Fig. 2). There was no significant inhibition of the uptake of the labelled precursor. Other inhibitors of the synthesis or secretion of cellulose or matrix polysaccharides, such as monensin (Brummel and Hall 1985) or cytochalasin D (Shannon et aI., 1984) inhibited incorporation into both !3-glucans to the same extent (Table 1). It has also been proposed that (1-3)-!3-glucanases could be involved in the metabolism of callose, either by hydrolysis or by transglucosylation (Bucheli et aI., 1985, 1987). Inhibitors of 13glucosidase or !3-glucanase, such as nojirimycin, I-deoxynojirimycin and N-methyl-1-deoxy-nojirimycin at 1 mM, all inhibited the incorporation into both !3-glucans, but nonspecifically (Table 1). Only the results obtained with DCB merited further study. Carpita and Delmer (1981) investigated the carbon flow from labelled glucose in ovule-grown cotton fibres and concluded that UDP-glucose was probably the precursor of both callose and cellulose. We have now examined the effect of DCB on some of the enzymes involved in the metabolism of low-molecular-weight carbohydrates of cotton fibre and its effect on the level of radioactivity in various sugar pools. The fact that DCB gives rise to a specific inhibition of cellulose synthesis, but has little effect on that of callose, should lead to an accumulation of radioactivity in some of the pools, e. g. in UDP-glucose.

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Table 1: The influence of various substances on the incorporation of [U)4C]glucose into iJ-glucans of the aerial fibres of cotton ovules cultured in vitro. The fibres were normally pretreated for 1 h before adding [U)4C]glucose (final concentration 5 mM, 296MBq/mmol) to the culture medium containing 60 mM pentaerythritol. The fibres were recovered after 1 h of incubation and exhaustively extracted with hot 80 % methanol. The callose in the fibre residue was solubilised in hot DMSO and the radioactivity in the DMSOsoluble (= callose) and the DMSO-insoluble (= cellulose) material counted.

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Concentration

% Total incorporation*

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At a concentration of 350 J.tM, DCB had no effect on the activity of uridine diphosphoglucose pyrophosphorylase, hexokinase, phosphoglucomutase, glucose 6-phosphate dehydrogenase, ,B-glucosidase, or exo-(1-3)-,B-glucanase in extracts from plant-grown cotton fibres (not shown). No inhibition was observed on the activity of (1-3 )-,B-glucan synthase in a microsomal membrane pellet derived from a homogenate of plant-grown cotton fibres. Some of the more important sugar pools were examined in the pulse-chase experiment described above. The sugars which could be solubilised in boiling 80 % ethanol were examined by HPLC. The sugar phosphate pool (glucose 6phosphate, fructose 6-phosphate and glucose 1-phosphate, present in ratio 8: 5: 1) was not resolved on HPLC nor were the individual UDP-sugars. The latter were isolated by selective adsorption on active charcoal followed by purification on DEAE-cellulose as described by Carpita and Delmer (1981). Acid hydrolysis followed by thin layer chromatography showed that UDP-glucose was the main sugar nucleotide (cf Carpita and Delmer, 1981), and at the beginning of the chase constituted at least 75 % of the radioactivity in the UDP-sugar pool. The radioactivity in the sugar pools was determined and the individual metabolites were estimated enzymatically. However, throughout the experiment the amounts of the individual metabolites did not change significantly.

DCB had no significant effect on the uptake of radioactivity from the incubation medium, but the amount of radioactivity in the pools of the fibres pre-treated with DCB was much higher (Fig. 6). The radioactivity in each of the pools was at a maximum at the end of the pulse and during the period of chase the radioactivity in each pool diminished regularly and in parallel for the fibres in the presence and absence of DCB.

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Discussion The experimental system described permits the feeding in vitro of radioactive precursors to intact cotton fibre cells, with little or no perturbation of the cells, and may be compared to the labelling experiments previously carried out in vivo (Meier et al., 1981), i.e. labelling of intact cotton plants with 14C02 and feeding [14C]sucrose to the petioles of cotton fruits. In all of these experimental systems, upon short-term feeding (several hours), the ratio of labelled cellulose to callose formed is about 2: 1. The use of cotton ovule cultures permits easy modification of the experimental conditions and thus more reliable pulse-chase experiments. The differences obtained for the incorporation into the fibre cell wall ,B-glucans from glucose, fructose and sucrose may be attributed to differences in their transport velocity

490

Y. FRANCEY, J. P. JAQUET, S. CAIROLI, A. J. BUCHALA, and H. MEIER

or to the sizes of their pools in the cell. Compared to glucose, the labelled sucrose is probably more readily transported to the vacuole where it would be highly diluted while the fructose is probably directed more readily to glycolysis. Fructokinase, ATP-phosphofructokinase, and pyrophosphate-phosphofructokinase (fructose 2,6-bisphosphatedependent) activities were found to be significant in cotton fibre homogenates, d. Huber and Akazawa (1986). The structure of the callose, which was almost completely solubilized from the fibres by treatment with hot DMSO, is quasi identical with that previously reported for the endogenous glucan in cotton fibres (Huwyler et aI., 1978; Meier et al., 1981), with the product synthesised from UDP-glucose by membrane fractions and detached cotton fibres (Heiniger and Delmer 1977) and with the product obtained on feeding sucrose to the petioles of cotton fruits (Meier et aI., 1981) or to isolated seed clusters (Pillonel and Meier, 1985). Such callose appeared uniformly labelled since the radioactivity associated with each structural feature was similar to the molar ratio of the structural features. On the other hand, no rigorous characterisation was made of the material insoluble in hot DMSO, but it was found to be chemically indistinguishable from cellulose. The labelled material in such a residue behaved like cellulose and was clearly (1 ..... 4)-{l-glucan. However, neither the degree of polymerisation nor the distribution of radioactivity were determined for the labelled product. This study was carried out in order to establish the role, if any, of synthesis of callose, concomitant with that of cellulose. Maltby et aI. (1979) found no evidence for turnover of callose in submerged fibres of cotton ovules grown in vitro. The results reported here, both concerning long-term continous feeding and the pulse-chase experiments carried out with the aerial fibres grown in vitro, show that callose does indeed undergo turnover. Exo-( 1.....3)-{l-glucanase and {l-glucosidase activities have been found and partially characterised in the cell wall of plant-grown cotton fibres (Bucheli et al., 1985). They are also present in the ovule culture fibres and in the culture medium (unpublished results). The callose in isolated cell wall fragments of plant-grown cotton fibres has been shown to undergo degradation by enzymes present in wall preparations (Bucheli et aI., 1987). Such degradation was low at the primary cell wall stage of development and increased rapidly (about 10-fold) at the beginning of secondary wall formation. The callose may be hydrolysed in vivo to produce glucose which in turn is partly used for the synthesis of cellulose. Nojirimycin inhibits not only {l-glucanase activity and hydrolysis of endogenous callose (Bucheli et aI., 1985, 1987), but also incorporation into the fibre cell wall {lglucans (Table 1). However, the latter inhibition did not produce the expected change in the ratio of callose to cellulose synthesised, so that no conclusion can be drawn. As reported previously (Montezinos and Delmer, 1980; Pillonel and Meier, 1985), DCB at 10 p.M gave rise to an inhibition in the incorporation of radioactivity into cellulose. Only slight inhibition of incorporation into callose was observed. This is contrary to the statement of Montezinos and Delmer (1980), but a close examination of their results shows that the reported inhibition was not pronounced. In the work currently reported only glucans were found to be la-

belled, whereas Montezinos and Delmer (1980) found about 30 % incorporation into non-glucan polysaccharides. The differences in the results could be explained by the fact that the submerged fibres studied by these workers are inevitably contaminated with callus tissue. The callus tissue has a composition akin to that of the primary cell wall of the fibres and incorporation of radioactivity from [14C]glucose into all the polysaccharides of the callus, including callose, occurs (unpublished results). Uridine diphosphoglucose, the probable precursor in the synthesis of cellulose, has been shown to undergo turnover in pulse-chase experiments with ovule-grown fibres (Carpita and Delmer, 1981). Our results confirm the turnover of UDP-glucose. The diagram for the metabolism of carbohydrates by these authors makes no mention of fructose, which is a major constituent of the non-structural carbohydrates of the cotton fibre Oaquet et aI., 1982). Radioactivity from fructose is incorporated into the {l-glucans of the fibre cell wall and several enzymes implicated in the metabolism of fructose are quite active in the fibre symplast. Nevertheless, the precursor role of UDP-glc is not disputed. The presence of DCB in the incubation medium during pulse-chase experiments did not have a significant effect on the kinetics of incorporation of radioactivity into callose (see Figs. 2 and 4) while the synthesis of cellulose was clearly inhibited. Similarly, DCB had no significant effect on the synthesis of callose in detached cotton fibres (unpublished observation). On the other hand, the radioactivity in the glucose, fructose and sucrose pool, the sugar phosphate (glucose 6-phosphate, glucose 1-phosphate and fructose 6-phosphate) pool, and the UDP-sugar (principally UDP-glucose) pool was several fold higher at the end of the [14C]glucose pulse. Such an effect was apparently not due to an increase in uptake of [14C]glucose from the incubation medium. The fact that none of the enzymes commonly involved in the metabolism of these pools was inhibited by DCB in vitro suggests that the DCB may act at the plasmalemma itself, probably upon the cellulase synthase complex. Delmer et aI. (1987) have recently reported that cotton fibres contain an 18 kD polypeptide which binds DCB. Such a polypeptide could playa role in the regulation of cellulose synthesis. The mechanism for synthesis of cellulose is still far from clear. The results reported here support the idea that callose may function as an intermediate in the synthesis of cellulose, via hydrolytic breakdown and reuse of the glucose residues, but no evidence has been obtained to show that callose is an obligate intermediate. Acknowledgements The authors wish to thank Mrs Y. Jenny for help in producing the cotton ovule cultures and Drs N. Carpita and U. Ryser for helpful discussions. This work was supported by the Swiss National Science Foundation.

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Biosynthesis of /3-glucans in cotton fibres BRUMMELL, D. A. and J. L. HAu.: The role of cell wall synthesis in sustained auxin-induced growth. Physiol. Plant. 63, 406-412 (1985). BUCHAlA, A. J. and H. MEIER.: Biosynthesis of /3-glucans in growing cotton (Gossypium arboreum L. and Gossypium hirsutum L.) fibres. In: Biochemistry of plant cell walls, pp. 221-241, BRETT, C. T. and HILLMAN, J. R., eds. Cambridge University Press, Cambridge (1985). BUCHEU, P., M. DOll, A. J. BUCHALA, and H. MEIER: /3-Glucanases in developing cotton (Gossypium hirsutum L.) fibres. Planta 166, 530 - 536 (1985). BUCHEU, P., A. J. BUCHAlA, and H. MEIER: Autolysis in vitro of cotton (Gossypium hirsutum L.) fibre cell walls. Physiol. Plant. 70, 633-638 (1987). CANEVSCINI, G., D. FRACHEBOUD, and H. MEIER: Fractionation and identification of cellulases and other extracellular enzymes produced by Sporotrichum (Chrysosporium) thermophile during growth on cellulose or cellobiose. Can. J. Microbiol. 29, 1071-1080 (1983).

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