Planta 149,306-312(1980)
P l a n t a 9 by Springer-Verlag 1980
Glucan Synthesis by Intact Cotton Fibres Fed with Different Precursors at the Stages of Primary and Secondary Wall Formation Christian Pillonel, Antony J. Buchala, and Hans Meier* Institut de Biologie v~g~taleet de Phytochimie, Universit~ de Fribourg, 3, Rue Albert Gockel, CH-1700 Fribourg, Switzerland
Abstract. Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 gM were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo-/~-l,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight *
To whom requests for reprints should be addressed
Abbreviations: UDPG=uridinediphosphoglucose; GDPG=guan-
osinediphosphoglucose; HEPES = N-2-hydroxyethylpiperazine-N'2-ethansulphonic acid; DMSO=dimethyl-sulfoxide; DNP=2,4dinitrophenol; DPA =days post anthesis
0032-0935/80/0149/0306/$ 01.40
material was found in cellulose and at least 2/3 in /%l,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation fl-l,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.
Key words: Callose - Cellulose - Cell wall - Fibre (cotton) - Glucan synthesis - Gossypium.
Introduction A number of investigations have been reported over the last twenty years on the synthesis in vitro of/~glucans by particulate cell fractions of higher land plants using nucIeoside diphosphate gIucose substrates (Feingold etal. 1958; Barber etal. 1964; Brummond and Gibbons 1965; Villemez et al. 1967; Flowers et al. 1968; Ordin and Hall 1968; Clark and Villemez 1972; Delmer etal. 1977; Helsper et al. 1977). The sources of the particulate fractions were mainly homogenates of tissues or cells at the stage of primary wall formation. Glucans containing mainly/~-l,3-1inkages (callose-type), mainly/3-1,4-linkages (cellulose-type), or /3-1,3- and /~-l,4-1inkages (cereal glucan-type) were diversely claimed to by synthesized by these systems. When UDPG was used as precursor the proportions of the two types of linkages depended on the concentration of the nucleotide sugar in the incubation medium (Ordin and Hall 1968; P6aud-Leno~l and Axelos 1970; Smith and Stone 1973; Van der Woude et al. t974; Raymond et al.
C. Pillonel et al. : Glucan Synthesis by Cotton Fibres
1978). Callose, a p-l,3-1inked glucan, is known to be formed in many cases when plant tissues are damaged. It is therefore not surprising that large amounts of ~-l,3-glucosidic linkages are usually formed in experiments in vitro with homogenates or damaged cells. In order to obtain a better understanding of cellulose synthesis it seems evident that one should work with undamaged cells or tissues in vivo. The present paper reports results obtained from experiments in vivo with cotton fibres at the stages of primary and secondary wall formation in which the radioactive precursors were fed to the exterior of the cells.
Materials and Methods Plant Material and Reagents'. Cotton plants (Gossypium arboreum L.) were grown in a greenhouse at a temperature of 25 to 30~ C during the day and 18 to 22~ at night. Fruits were harvested from plants eight to ten years old. Under the described cultivation conditions the fibres reached maturity in about 60 days. [U-14C]Glucose (7.46.109 Bq retool- 1), [U_14C]sucrose (1.36. 10 l~ Bq retool-l), UDP[U-14C]glucose (1.1-10 l~ Bq retool-l), and GDP[U-14C]glucose (9.2.109 Bq retool- 1) were obtained from ICN (Irvine, Calif., USA). Exo-fl-1,3-glucanase (/L1,3-glucan glucohydrolase, EC 3.2.1.58) was prepared from a culture filtrate of Basidiomycete sp. QM 806. The fungus was cultivated on the liquid medium proposed by Reese and Mandels (1959), containing 0.3% glucose as a carbon source, in which urea was replaced by Neopeptone (Difco). The culture was incubated at 25~ C while stirring. After six days the culture medium was filtered and then dialyzed using a hollow fibre system with a nominal molecular retention limit of L000 D (Amicon Corp., Lexington, Mass., USA). The enzyme was partially purified by fractionation with ammonium sulphate, according to Huotari et al. (1968). The freeze-dried enzyme preparation released 80 ~tmole of glucose per rain per mg protein when incubated at 40 ~ C with laminaran (0.5 mg ml- 1) in 0.01 M sodium citrate buffer (pH 5). Glucose was determined with glucose oxidase (GOD-Perid method, Boehringer, Mannheim, FRG). The enzyme preparation also had some fi-glucosidase activity. When incubated with amorphous cellulose under the same conditions described for laminaran, the number of glucose equivalents released was about 1,000 times smaller. Feeding of Radioactive Precursors. Fruit capsules were harvested at different times after anthesis. The capsules, usually containing three carpells, were immediately dissected and the intact seed clusters (each containing six or seven seeds enveloped in a network of their fibres) were individually removed from each of the three locules. The seed cluster from one locule was then incubated in a filter test tube (inner diameter 21 ram) with a side arm near the upper end which could be connected to a vacuum pump. The incubation medium (5 ml) contained HEPES buffer (20 raM, pH 7.5), Triton X-100 (0.01%), and radioactive precursor (5, 200, or 1~000 btM). In most experiments the radioactivity was 3.8- 104 or 18.9.104 Bq per ml incubation medium. To improve impregnation of the seed clusters and adhering fibres with the liquid medium, the test tube was evacuated carefully three times fro" approx. 20 s by connection to a water pump. Incubation was in an open tube at 30~ C for one hour with intermediate gentle agitation by hand.
307
Extraction of Low Molecular Weight Substances'. At the end of each incubation the seed clusters were superficially rinsed with ice-cold water, the fibres quickly detached from the seeds with forceps and transferred to hot 80% (v/v) methanol. They were then coarsely cut with scissors, homogenized in liquid nitrogen, and exhaustively extracted with hot 80% methanol, with intermediate filtration, until no more radioactive material could be solubilized. The fibres were suspended in water and freeze dried. An aliquot of the methanol-extracted freeze-dried fibres, hereafter referred to as the methanol-extracted fibres, was counted in the scintillation counter.
Determination of the Radioactivity in the Soluble and in the Insoluble High Molecular Weight Products Formed. Two methods were used to extract the non-cellulosic glucans from the fibres treated as described above - both methods gave essentially similar results: (i) An aliquot of the fibres (about 50 rag) was extracted on a shaker for 6 h with a solution (50 ml) of NaOH (17.5%) containing H3BO~ (4%) under a nitrogen atmosphere at room temperature. The suspension was then filtered through fibreglass paper (Whatman GF/A), the residue washed with acetic acid (40%) followed by water and then freeze dried. The combined filtrate and washings were neutralized with acetic acid (40%) and dialyzed against water in the cold. The radioactivity in aliquots of the residue and extract was measured. (ii) An aliquot of the fibres (100 to 200 mg) was treated three times with DMSO (30 ml) for 30 min at 120~ in the autociav.e. After each treatment the fibres were recovered by filtration and the final residue was washed with water. The radioactivity in the final residue and the combined extracts was measured.
Enzyme
Treatments. Homogenized methanol-extracted fibres (50 rag) were suspended in 0.01 M sodium citrate buffer (pH 5.0, 10 ml) and after addition of 40 gg exo-fi-l,3-glucanase incubated for 6 h at 40~ on a shaker. The suspension was then filtered and the insoluble residue incubated for another 2 h in fresh glucanase-containing incubation medium. The insoluble residue was recovered by filtration and the solubilized material pooled and deionized. Aliquots of each were counted in the scintillation counter and samples of the latter examined by paper and thin layer chromatography. The high molecular weight material contained in the alkali or DMSO extracts from the fibres was also treated with the exo-fi-l,3-glucanase and examined as described above. Acid Hydrolysis, Radio-Chromatography of the Products and Radio Counting. Acid hydro~sis of the methanol-extracted fibres was carried out with 72% and 4% H2SOr according to Saemen et al. (1954). Paper chromatography was on Schleicher and Schtill No 2043b paper and thin layer chromatography on Kieselgel 60 (Merck, Darmstadt, FRG) using the following solvents: (A) propanol- 1 : ethyl acetate: water (7: I : 2, v/v) for separation of glucose, laminaribiose, cellobiose and laminaritriose; (B) ethyl acetate: pyridine:water (2: 1:2, v/v~ upper phase) for separation of mono- and oligosaccharides; (C) ethyl acetate: pyridine: water (8 : 2 : 1, v/v) for monosaccharides; (D) acetone: water (87 : 13, v/v) with two developments at 40 ~ C and intermediate drying for separation of glucose, laminaribiose and cellobiose. Detection was either with alkaline AgNO3 (Trevelyan 1950) or with ~-naphthol/conc. H2SO4 (Stahl 1969) where appropriate. Localization of radioactive spots on chromatograms was done with a radioscanner LB 2723 (Berthold, Mfinich, FRG). Measurement of the radioactivity in freeze-dried homogenates of fibres was carried out by swelling a sample of the fibres in water (5 ml) followed by the addition of Lumagel (10 ml; Fakola AG, Basle, Switzerland) and thorough mixing so as to form a stable homogeneous gel. Enzymic digests, alkaline or DMSO extracts, after removal of salts or DMSO, were counted in a mixture of water (5ml) and Lumagel (10ml). Scintillation counting was done with a Packard Tri-Carb 2425 spectrometer.
308
C. Pillonel et al. : Glucan Synthesis by Cotton Fibres
60000-
Table 1, Incorporation of [i4C]glucose from different precursors into the 80% methanol-insoluble residues of cotton fibres at two
0.(54860)
different stages of development. Feeding was to seed clusters with intact adhering fibres during 1 h at 30 ~ C
50000-
Radioactive" precursors fed
Primary wall fibres b : pmol of glucose incorporated per fibres of one seed
UDPG UDPG GDPG GDPG
0.8 3,666.0 1.5 92.5
16.7 5,764.0 2.6 161.7
20.9 1.6 1.7 1.8
Glucose 5 ~tM d Glucose 1,000 g M d
0.3 52.0
8.3 1,197.0
27.6 23.0
Sucrose 5 ]-tMd Sucrose 1,000 ~tM d
0.6 e 80.0 e
40000-
== 3oooo0
20000-
g
e ( 50 O) 10000.
24
do
~
d2
d8
fibre age in days Fig. ]. Incorporation o f radioactive sucrose into the 80% methanolinsoluble residues of cotton fibres of different ages, The incubation m e d i u m contained 0.2 m M sucrose (18.9.104 Bq ml 1), the incubation time was 1 h and the temperature 30 ~ C
Results
Incorporation of Sucrose into Products of High Molecular Weight by Fibres of Different Ages. Cotton fruits were harvested 24, 30, 36, 42, and 48 days post anthesis (DPA). The fibres 24 DPA had only primary walls, whereas after 30 days secondary wall formation had just started. The seed clusters of single fruit locules were incubated with radioactive sucrose for 1 h and the radioactivity in the methanol-extracted fibres, i.e., incorporated into products of high molecular weight, was measured. Figure 1 shows that for this series of experiments, the incorporation rate was highest for the fibres 36 DPA and was significant throughout the period of secondary wall formation, whereas at 24 DPA the rate of incorporation was about 300 times less than the maximum. However, it was also observed that the age at which the maximum incorporation rate occurred varied during the growth season and depended to some extent on the lighting conditions in the greenhouse.
Comparison of the Incorporation of Different Radioactive Precursors into Products of High Molecular Weight at the Stages of Primary and Secondary Wall Formation. For the primary wall fibres it can be seen
5 gM 1,000 g M 5 gM 1,000 llM
Secondary wall fibres ~ pmol of glucose incorporated per fibres of one seed
81.2 ~ 11,061.0 e
Ratio of incorporation of secondary to primary wall fibres
135 138
Uniformety Iabelled in the sugar moieties b Age: 22 days post anthesis c Age: 35 days post anthesis a Since the apoplast of the fibres already naturally contains some glucose and sucrose, the real concentration and hence also the incorporation of these sugars is higher than indicated e Calculated for the glucose moiety of the sucrose molecule. The fructose moiety, however, might also have contributed to the values obtained since uniformely labelled sucrose was used
from Table 1 that the glucose incorporation from all of the precursors was relatively low and rather similar except for U D P G at high concentrations. F r o m U D P G at 1,000 gM over 4,000 times more glucose was incorporated than from U D P G at 5 txM, whereas the precursor concentrations differ only by a factor of 200. N o such "concentration-effect" was observed with G D P G , glucose, or sucrose. For the secondary wall fibres, Table 1 shows that sucrose was by far the best precursor at high or low concentrations and the rate of incorporation in this series of experiments was almost 140 times that for primary wall fibres. Incorporation from U D P G behaved completely differently - at high concentrations it was not even twice that for primary wall fibres. The same was true for G D P G . Incorporation from glucose was more like that from sucrose although the rate for secondary wall fibres compared to that for primary wall fibres was not so drastically increased. The increase in the incorporation rate of sucrose seems to parallel the increase in the rate of cellulose synthesis for secondary wall fibres observed by Huwyler et al. (1979). In order to determine whether or not sucrose and U D P G are competitive precursors, they were fed simultaneously and their incorporations were corn-
C. Pillonel et al. : Glucan Synthesis by Cotton Fibres Table 2. Influence of UDPG on the incorporation of sucrose, and vice versa, into the 80% methanol-insoluble residues of cotton fibres. The feeding conditions were the same as those stated in Table 1. Incorporations are given in % of those without "competitor" (= 100%) Radioacitve precursor
Sucrose 5 gM UDPG 5 gM
"Competitor" (non-radioactive)
UDPG 1,000 btM Sucrose 1,000 gM
Incorporation of radioactivity by primary wall fibres
Incorporation of radioactivity by secondary wall fibres
91% 70%
97% 99%
Table 3. Influence of KCN and DNP on incorporation of radioactivity from UDPG and sucrose into the 80% methanol-insoluble residues of cotton fibres Radioactive precursor and inhibitors
Incorporation by primary wall fibres
Incorporation by secondary wall fibres
Sucrose 1 mM Sucrose 1 m M + K C N 1 mM Sucrose 1 m M + D N P 0.5 mM
100% 23% 18%
100% 52% 27%
UDPG 1 mM UDPG 1 m M + K C N 1 mM UDPG 1 m M + D N P 0.5raM
100% 100% 90%
100% 91% 93%
pared to those obtained when each was fed individually. Table 2 shows that there is no competition between the two, indicating that their metabolic pathways are well separated when they are fed to the exterior of the cells. Inhibitors of respiratory ATP-formation such as KCN and DNP strongly inhibited sucrose incorporation but did not have much influence on that from UDPG (see Table 3). In addition, it was observed that mechanical damage (disruption of the fibres) resulted in an almost complete loss of the ability of the secondary wall fibres to incorporate glucose from sucrose and that there was no such effect on the incorporation from UDPG, whose incorporation was even improved.
Study of the Glucans Formed by Secondary Wall Fibres from Radioactive UDPG and Sucrose, respectively. Total acid hydrolysis of the freeze-dried, methanolextracted, secondary wall fibres fed with either [Ul~C]sucrose or UDP[U-lgC]glucose and paper chromatography (solvent C) gave glucose as the only radioactive monosaccharide, showing in both cases that the high molecular weight products were essentially glucans. The bulk of the non-cellulosic polysaccharides (and eventually glucoproteins) was extracted
309 Table 4. Percentage of radioactivity not solubilised with alkali or exo-/3-1,3-glucanase from 80% methanol treated secondary wall fibres, which had previously been fed with either radioactive UDPG or sucrose Radioactive substrates fed to secondary wall cotton fibres UDPG 5 gM
UDPG 1,000 gM
Sucrose 5 gM
Sucrose 1,000 btM
Residual radioactivity in the alkali extracted fibres
28%
7%
25%
29%
Residual radioactivity in the /~-1,3-glucanase treated fibres
25%
4%
30%
29%
from a sample of the methanol-extracted fibres with an alkaline solution containing 17.5% NaOH and 4% H3BO 3. Table 4 shows the percentage of the radioactivity remaining in the insoluble residues from secondary wall fibres fed with radioactive UDPG or sucrose, respectively (see also Table 1 for the total incorporation into high molecular weight products). Assuming these insoluble residues are essentially cellulose, the results show that from UDPG at 1,000 btM only a very small percentage of glucose was incorporated into cellulose, whereas from UDPG at 5 gM and from sucrose at both concentrations, between 25 and 29% glucose was incorporated into cellulosic glucan. Some of the alkali extracts were degraded with exo-fi-l,3-glucanase from Basidiomycete sp. QM 806. Thin layer chromatography (solvent D) of the products showed that nearly all of the radioactivity was confined to glucose, indicating that the radioactivity in the extracts came from fi-l,3-glucans. Direct exhaustive treatment of the methanol-extracted fibres with the fl-1,3-glucanase gave results which were qualitatively and quantitatively similar to those obtained by alkaline extraction (Table 4). Paper chromatography (solvent A) of the degradation products obtained after enzymic treatment of the methanol-extracted fibres fed with either radioactive UDPG at 5 and 1,000txM or sucrose at 5 and 1,000 laM gave similar results: two radioactive components were always obtained chromatographically identical to glucose and laminaritriose, the latter however in trace quantities. To study whether the formation of fi-l,3-glucans is restricted to certain ages of the secondary wall fibres, the proportion formed by fibres at various ages from 30 to 48 DPA was determined. The "noncellulosic" glucans were exhaustively extracted, with DMSO at 120~ C in the autoclave, from the methanol-
310
C. Pillonel etal. : Glucan Synthesis by Cotton Fibres
4O000
-
I~
o
P l a n t a 9 by Springer-Verlag 1980
Glucan Synthesis by Intact Cotton Fibres Fed with Different Precursors at the Stages of Primary and Secondary Wall Formation Christian Pillonel, Antony J. Buchala, and Hans Meier* Institut de Biologie v~g~taleet de Phytochimie, Universit~ de Fribourg, 3, Rue Albert Gockel, CH-1700 Fribourg, Switzerland
Abstract. Seed clusters of individual locules from fruit capsules of Gossypium arboreum L. with adhering intact fibres were fed with radioactive uridinediphosphoglucose (UDPG), guanosinediphosphoglucose (GDPG), glucose and sucrose. The incorporation into high molecular weight glucans of the fibres was studied. For primary wall fibres, UDPG at 1 mM was by far the best precursor, whereas sucrose was the best precursor for secondary wall fibres. No competition was observed between the incorporation of glucose from UDPG and from sucrose when the two were fed simultaneously to secondary wall fibres, indicating that their metabolic pathways are well separated when they are fed from the apoplast. Inhibitors of respiratory ATP-formation strongly inhibited incorporation of sucrose but not that of UDPG. Sucrose incorporation was studied at five different stages of development of the cotton fibres. At the stage of most intense secondary wall formation the incorporation rate was about 300 times that during primary wall formation (24 days post anthesis (DPA)). Incorporation from 1 mM UDPG or GDPG by secondary wall fibres (35 DPA) was less than twice that of primary wall fibres (22 DPA), indicating that the two sugar nucleotides are not readily used as precursors for secondary wall cellulose when they are fed to the exterior of intact cells. The high molecular weight non-cellulosic glucans formed from UDPG and sucrose at 5 and 1,000 gM were solubilized in strongly alkaline solutions or dimethyl-sulfoxide (DMSO) and were partially characterized by degradation with an exo-/~-l,3-glucanase. After feeding for one hour, at most 1/3 of the radioactivity in high molecular weight *
To whom requests for reprints should be addressed
Abbreviations: UDPG=uridinediphosphoglucose; GDPG=guan-
osinediphosphoglucose; HEPES = N-2-hydroxyethylpiperazine-N'2-ethansulphonic acid; DMSO=dimethyl-sulfoxide; DNP=2,4dinitrophenol; DPA =days post anthesis
0032-0935/80/0149/0306/$ 01.40
material was found in cellulose and at least 2/3 in /%l,3-glucan. The proportions varied little for fibres in the age range of 30 to 48 DPA when sucrose was the precursor although the total incorporation varied by a factor of about four. The fact that at all stages of secondary wall formation fl-l,3-glucan is synthesized at a very high rate, but that the total amount in the cell wall does not exceed 2% in the later stages of wall formation, can be interpreted in terms of a high turnover of this polysaccharide if it is assumed that wound effects are negligible in the system under study.
Key words: Callose - Cellulose - Cell wall - Fibre (cotton) - Glucan synthesis - Gossypium.
Introduction A number of investigations have been reported over the last twenty years on the synthesis in vitro of/~glucans by particulate cell fractions of higher land plants using nucIeoside diphosphate gIucose substrates (Feingold etal. 1958; Barber etal. 1964; Brummond and Gibbons 1965; Villemez et al. 1967; Flowers et al. 1968; Ordin and Hall 1968; Clark and Villemez 1972; Delmer etal. 1977; Helsper et al. 1977). The sources of the particulate fractions were mainly homogenates of tissues or cells at the stage of primary wall formation. Glucans containing mainly/~-l,3-1inkages (callose-type), mainly/3-1,4-linkages (cellulose-type), or /3-1,3- and /~-l,4-1inkages (cereal glucan-type) were diversely claimed to by synthesized by these systems. When UDPG was used as precursor the proportions of the two types of linkages depended on the concentration of the nucleotide sugar in the incubation medium (Ordin and Hall 1968; P6aud-Leno~l and Axelos 1970; Smith and Stone 1973; Van der Woude et al. t974; Raymond et al.
C. Pillonel et al. : Glucan Synthesis by Cotton Fibres
1978). Callose, a p-l,3-1inked glucan, is known to be formed in many cases when plant tissues are damaged. It is therefore not surprising that large amounts of ~-l,3-glucosidic linkages are usually formed in experiments in vitro with homogenates or damaged cells. In order to obtain a better understanding of cellulose synthesis it seems evident that one should work with undamaged cells or tissues in vivo. The present paper reports results obtained from experiments in vivo with cotton fibres at the stages of primary and secondary wall formation in which the radioactive precursors were fed to the exterior of the cells.
Materials and Methods Plant Material and Reagents'. Cotton plants (Gossypium arboreum L.) were grown in a greenhouse at a temperature of 25 to 30~ C during the day and 18 to 22~ at night. Fruits were harvested from plants eight to ten years old. Under the described cultivation conditions the fibres reached maturity in about 60 days. [U-14C]Glucose (7.46.109 Bq retool- 1), [U_14C]sucrose (1.36. 10 l~ Bq retool-l), UDP[U-14C]glucose (1.1-10 l~ Bq retool-l), and GDP[U-14C]glucose (9.2.109 Bq retool- 1) were obtained from ICN (Irvine, Calif., USA). Exo-fl-1,3-glucanase (/L1,3-glucan glucohydrolase, EC 3.2.1.58) was prepared from a culture filtrate of Basidiomycete sp. QM 806. The fungus was cultivated on the liquid medium proposed by Reese and Mandels (1959), containing 0.3% glucose as a carbon source, in which urea was replaced by Neopeptone (Difco). The culture was incubated at 25~ C while stirring. After six days the culture medium was filtered and then dialyzed using a hollow fibre system with a nominal molecular retention limit of L000 D (Amicon Corp., Lexington, Mass., USA). The enzyme was partially purified by fractionation with ammonium sulphate, according to Huotari et al. (1968). The freeze-dried enzyme preparation released 80 ~tmole of glucose per rain per mg protein when incubated at 40 ~ C with laminaran (0.5 mg ml- 1) in 0.01 M sodium citrate buffer (pH 5). Glucose was determined with glucose oxidase (GOD-Perid method, Boehringer, Mannheim, FRG). The enzyme preparation also had some fi-glucosidase activity. When incubated with amorphous cellulose under the same conditions described for laminaran, the number of glucose equivalents released was about 1,000 times smaller. Feeding of Radioactive Precursors. Fruit capsules were harvested at different times after anthesis. The capsules, usually containing three carpells, were immediately dissected and the intact seed clusters (each containing six or seven seeds enveloped in a network of their fibres) were individually removed from each of the three locules. The seed cluster from one locule was then incubated in a filter test tube (inner diameter 21 ram) with a side arm near the upper end which could be connected to a vacuum pump. The incubation medium (5 ml) contained HEPES buffer (20 raM, pH 7.5), Triton X-100 (0.01%), and radioactive precursor (5, 200, or 1~000 btM). In most experiments the radioactivity was 3.8- 104 or 18.9.104 Bq per ml incubation medium. To improve impregnation of the seed clusters and adhering fibres with the liquid medium, the test tube was evacuated carefully three times fro" approx. 20 s by connection to a water pump. Incubation was in an open tube at 30~ C for one hour with intermediate gentle agitation by hand.
307
Extraction of Low Molecular Weight Substances'. At the end of each incubation the seed clusters were superficially rinsed with ice-cold water, the fibres quickly detached from the seeds with forceps and transferred to hot 80% (v/v) methanol. They were then coarsely cut with scissors, homogenized in liquid nitrogen, and exhaustively extracted with hot 80% methanol, with intermediate filtration, until no more radioactive material could be solubilized. The fibres were suspended in water and freeze dried. An aliquot of the methanol-extracted freeze-dried fibres, hereafter referred to as the methanol-extracted fibres, was counted in the scintillation counter.
Determination of the Radioactivity in the Soluble and in the Insoluble High Molecular Weight Products Formed. Two methods were used to extract the non-cellulosic glucans from the fibres treated as described above - both methods gave essentially similar results: (i) An aliquot of the fibres (about 50 rag) was extracted on a shaker for 6 h with a solution (50 ml) of NaOH (17.5%) containing H3BO~ (4%) under a nitrogen atmosphere at room temperature. The suspension was then filtered through fibreglass paper (Whatman GF/A), the residue washed with acetic acid (40%) followed by water and then freeze dried. The combined filtrate and washings were neutralized with acetic acid (40%) and dialyzed against water in the cold. The radioactivity in aliquots of the residue and extract was measured. (ii) An aliquot of the fibres (100 to 200 mg) was treated three times with DMSO (30 ml) for 30 min at 120~ in the autociav.e. After each treatment the fibres were recovered by filtration and the final residue was washed with water. The radioactivity in the final residue and the combined extracts was measured.
Enzyme
Treatments. Homogenized methanol-extracted fibres (50 rag) were suspended in 0.01 M sodium citrate buffer (pH 5.0, 10 ml) and after addition of 40 gg exo-fi-l,3-glucanase incubated for 6 h at 40~ on a shaker. The suspension was then filtered and the insoluble residue incubated for another 2 h in fresh glucanase-containing incubation medium. The insoluble residue was recovered by filtration and the solubilized material pooled and deionized. Aliquots of each were counted in the scintillation counter and samples of the latter examined by paper and thin layer chromatography. The high molecular weight material contained in the alkali or DMSO extracts from the fibres was also treated with the exo-fi-l,3-glucanase and examined as described above. Acid Hydrolysis, Radio-Chromatography of the Products and Radio Counting. Acid hydro~sis of the methanol-extracted fibres was carried out with 72% and 4% H2SOr according to Saemen et al. (1954). Paper chromatography was on Schleicher and Schtill No 2043b paper and thin layer chromatography on Kieselgel 60 (Merck, Darmstadt, FRG) using the following solvents: (A) propanol- 1 : ethyl acetate: water (7: I : 2, v/v) for separation of glucose, laminaribiose, cellobiose and laminaritriose; (B) ethyl acetate: pyridine:water (2: 1:2, v/v~ upper phase) for separation of mono- and oligosaccharides; (C) ethyl acetate: pyridine: water (8 : 2 : 1, v/v) for monosaccharides; (D) acetone: water (87 : 13, v/v) with two developments at 40 ~ C and intermediate drying for separation of glucose, laminaribiose and cellobiose. Detection was either with alkaline AgNO3 (Trevelyan 1950) or with ~-naphthol/conc. H2SO4 (Stahl 1969) where appropriate. Localization of radioactive spots on chromatograms was done with a radioscanner LB 2723 (Berthold, Mfinich, FRG). Measurement of the radioactivity in freeze-dried homogenates of fibres was carried out by swelling a sample of the fibres in water (5 ml) followed by the addition of Lumagel (10 ml; Fakola AG, Basle, Switzerland) and thorough mixing so as to form a stable homogeneous gel. Enzymic digests, alkaline or DMSO extracts, after removal of salts or DMSO, were counted in a mixture of water (5ml) and Lumagel (10ml). Scintillation counting was done with a Packard Tri-Carb 2425 spectrometer.
308
C. Pillonel et al. : Glucan Synthesis by Cotton Fibres
60000-
Table 1, Incorporation of [i4C]glucose from different precursors into the 80% methanol-insoluble residues of cotton fibres at two
0.(54860)
different stages of development. Feeding was to seed clusters with intact adhering fibres during 1 h at 30 ~ C
50000-
Radioactive" precursors fed
Primary wall fibres b : pmol of glucose incorporated per fibres of one seed
UDPG UDPG GDPG GDPG
0.8 3,666.0 1.5 92.5
16.7 5,764.0 2.6 161.7
20.9 1.6 1.7 1.8
Glucose 5 ~tM d Glucose 1,000 g M d
0.3 52.0
8.3 1,197.0
27.6 23.0
Sucrose 5 ]-tMd Sucrose 1,000 ~tM d
0.6 e 80.0 e
40000-
== 3oooo0
20000-
g
e ( 50 O) 10000.
24
do
~
d2
d8
fibre age in days Fig. ]. Incorporation o f radioactive sucrose into the 80% methanolinsoluble residues of cotton fibres of different ages, The incubation m e d i u m contained 0.2 m M sucrose (18.9.104 Bq ml 1), the incubation time was 1 h and the temperature 30 ~ C
Results
Incorporation of Sucrose into Products of High Molecular Weight by Fibres of Different Ages. Cotton fruits were harvested 24, 30, 36, 42, and 48 days post anthesis (DPA). The fibres 24 DPA had only primary walls, whereas after 30 days secondary wall formation had just started. The seed clusters of single fruit locules were incubated with radioactive sucrose for 1 h and the radioactivity in the methanol-extracted fibres, i.e., incorporated into products of high molecular weight, was measured. Figure 1 shows that for this series of experiments, the incorporation rate was highest for the fibres 36 DPA and was significant throughout the period of secondary wall formation, whereas at 24 DPA the rate of incorporation was about 300 times less than the maximum. However, it was also observed that the age at which the maximum incorporation rate occurred varied during the growth season and depended to some extent on the lighting conditions in the greenhouse.
Comparison of the Incorporation of Different Radioactive Precursors into Products of High Molecular Weight at the Stages of Primary and Secondary Wall Formation. For the primary wall fibres it can be seen
5 gM 1,000 g M 5 gM 1,000 llM
Secondary wall fibres ~ pmol of glucose incorporated per fibres of one seed
81.2 ~ 11,061.0 e
Ratio of incorporation of secondary to primary wall fibres
135 138
Uniformety Iabelled in the sugar moieties b Age: 22 days post anthesis c Age: 35 days post anthesis a Since the apoplast of the fibres already naturally contains some glucose and sucrose, the real concentration and hence also the incorporation of these sugars is higher than indicated e Calculated for the glucose moiety of the sucrose molecule. The fructose moiety, however, might also have contributed to the values obtained since uniformely labelled sucrose was used
from Table 1 that the glucose incorporation from all of the precursors was relatively low and rather similar except for U D P G at high concentrations. F r o m U D P G at 1,000 gM over 4,000 times more glucose was incorporated than from U D P G at 5 txM, whereas the precursor concentrations differ only by a factor of 200. N o such "concentration-effect" was observed with G D P G , glucose, or sucrose. For the secondary wall fibres, Table 1 shows that sucrose was by far the best precursor at high or low concentrations and the rate of incorporation in this series of experiments was almost 140 times that for primary wall fibres. Incorporation from U D P G behaved completely differently - at high concentrations it was not even twice that for primary wall fibres. The same was true for G D P G . Incorporation from glucose was more like that from sucrose although the rate for secondary wall fibres compared to that for primary wall fibres was not so drastically increased. The increase in the incorporation rate of sucrose seems to parallel the increase in the rate of cellulose synthesis for secondary wall fibres observed by Huwyler et al. (1979). In order to determine whether or not sucrose and U D P G are competitive precursors, they were fed simultaneously and their incorporations were corn-
C. Pillonel et al. : Glucan Synthesis by Cotton Fibres Table 2. Influence of UDPG on the incorporation of sucrose, and vice versa, into the 80% methanol-insoluble residues of cotton fibres. The feeding conditions were the same as those stated in Table 1. Incorporations are given in % of those without "competitor" (= 100%) Radioacitve precursor
Sucrose 5 gM UDPG 5 gM
"Competitor" (non-radioactive)
UDPG 1,000 btM Sucrose 1,000 gM
Incorporation of radioactivity by primary wall fibres
Incorporation of radioactivity by secondary wall fibres
91% 70%
97% 99%
Table 3. Influence of KCN and DNP on incorporation of radioactivity from UDPG and sucrose into the 80% methanol-insoluble residues of cotton fibres Radioactive precursor and inhibitors
Incorporation by primary wall fibres
Incorporation by secondary wall fibres
Sucrose 1 mM Sucrose 1 m M + K C N 1 mM Sucrose 1 m M + D N P 0.5 mM
100% 23% 18%
100% 52% 27%
UDPG 1 mM UDPG 1 m M + K C N 1 mM UDPG 1 m M + D N P 0.5raM
100% 100% 90%
100% 91% 93%
pared to those obtained when each was fed individually. Table 2 shows that there is no competition between the two, indicating that their metabolic pathways are well separated when they are fed to the exterior of the cells. Inhibitors of respiratory ATP-formation such as KCN and DNP strongly inhibited sucrose incorporation but did not have much influence on that from UDPG (see Table 3). In addition, it was observed that mechanical damage (disruption of the fibres) resulted in an almost complete loss of the ability of the secondary wall fibres to incorporate glucose from sucrose and that there was no such effect on the incorporation from UDPG, whose incorporation was even improved.
Study of the Glucans Formed by Secondary Wall Fibres from Radioactive UDPG and Sucrose, respectively. Total acid hydrolysis of the freeze-dried, methanolextracted, secondary wall fibres fed with either [Ul~C]sucrose or UDP[U-lgC]glucose and paper chromatography (solvent C) gave glucose as the only radioactive monosaccharide, showing in both cases that the high molecular weight products were essentially glucans. The bulk of the non-cellulosic polysaccharides (and eventually glucoproteins) was extracted
309 Table 4. Percentage of radioactivity not solubilised with alkali or exo-/3-1,3-glucanase from 80% methanol treated secondary wall fibres, which had previously been fed with either radioactive UDPG or sucrose Radioactive substrates fed to secondary wall cotton fibres UDPG 5 gM
UDPG 1,000 gM
Sucrose 5 gM
Sucrose 1,000 btM
Residual radioactivity in the alkali extracted fibres
28%
7%
25%
29%
Residual radioactivity in the /~-1,3-glucanase treated fibres
25%
4%
30%
29%
from a sample of the methanol-extracted fibres with an alkaline solution containing 17.5% NaOH and 4% H3BO 3. Table 4 shows the percentage of the radioactivity remaining in the insoluble residues from secondary wall fibres fed with radioactive UDPG or sucrose, respectively (see also Table 1 for the total incorporation into high molecular weight products). Assuming these insoluble residues are essentially cellulose, the results show that from UDPG at 1,000 btM only a very small percentage of glucose was incorporated into cellulose, whereas from UDPG at 5 gM and from sucrose at both concentrations, between 25 and 29% glucose was incorporated into cellulosic glucan. Some of the alkali extracts were degraded with exo-fi-l,3-glucanase from Basidiomycete sp. QM 806. Thin layer chromatography (solvent D) of the products showed that nearly all of the radioactivity was confined to glucose, indicating that the radioactivity in the extracts came from fi-l,3-glucans. Direct exhaustive treatment of the methanol-extracted fibres with the fl-1,3-glucanase gave results which were qualitatively and quantitatively similar to those obtained by alkaline extraction (Table 4). Paper chromatography (solvent A) of the degradation products obtained after enzymic treatment of the methanol-extracted fibres fed with either radioactive UDPG at 5 and 1,000txM or sucrose at 5 and 1,000 laM gave similar results: two radioactive components were always obtained chromatographically identical to glucose and laminaritriose, the latter however in trace quantities. To study whether the formation of fi-l,3-glucans is restricted to certain ages of the secondary wall fibres, the proportion formed by fibres at various ages from 30 to 48 DPA was determined. The "noncellulosic" glucans were exhaustively extracted, with DMSO at 120~ C in the autoclave, from the methanol-
310
C. Pillonel etal. : Glucan Synthesis by Cotton Fibres
4O000
-
I~
o
