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Extracellular polysaccharides from suspension-cultured cells of Rubus fruticosus. Structure of a galactoglucomannan
Food Hydrocolloids vol.! no.5/6 pp.555 - 556, 1987
Extracellular polysaccharides from suspension-cultured cells of Rubus fruticosus. Structure of a galactoglucomannan G.Chambat, N.Cartier and J.P.Joseleau Centre de Recherches sur les Macromolecules Vegetales, B.P. 68, 38402 Saint-Martin d'Heres-Cedex, France Abstract. Carbohydrate analysis of the cell walls from suspension-cultured cells of Rubus fruticosus between day 10 and day 40 after inoculation revealed an overall increase of the net content of neutral sugars. However the composition of the extracellular polysaccharides showed only discrete variation. A galactoglucomannan containing galactose, glucose and mannose in the ratio 1.0: 1.4: 1.2 respectively, could be purified by barium hydroxide precipitation both in the extracellular medium and from the alkaline extract of the cell walls. Methylation analysis and 13C-NMR spectroscopy demonstrated that the extracellular form is the counterpart of the wall polysaccharide.
Introduction The extracellular medium of suspension-cultured cells is known to be rich in several types of water soluble polysaccharides. The extracellular polysaccharides (ECP) have been shown to have structures very much comparable to those of the cell wall polymers (CWP) (l). The mechanisms by which ECPs are released in the medium are not understood, neither is it known the possible roles that they may play in the cell culture. In the present work we analyzed the extracellular medium during Rubus fruticosus cell growth and compared it to the cell wall composition between days 10 and 40. We also report the isolation and structural determination of a typical galactoglucomannan from primary walls. Results Carbohydrate analysis of the cell walls at different ages and of the corresponding extracellular media revealed that all the sugars present in the cell wall are also found Table I. Changes in the composition of CWP and of ECP during the culture of Rifruticosus cells Uronic
Neutral
Neutral sugars (mol. %)
acids (wt %)
sugars (wt %)
Rhamnose
Fucose
dlO d 18 d 24 d 30 d 40
20.5 18.5 19.5 17.0 19.5
58.0 66.5 65.5 68.0 61.0
4 2.5 4 3.5 3
1.5 1.5 2 2.5 1.5
dlO d 18 d 24 d 30 d 40
28 30 27.5 27 27
50.5 45 43.5 49 35
6.5 4 3 4.5 4
5.5 4 3 3.5 4
Arabinose
CWP 19.5 19 16 20 12.5 ECP 21.5 17 13 19.5 IS
Xylose
Mannose
Galactose
Glucose
8.5 9.5 10.5 12 12.5
4 2.5 2.5 3 2.5
12 10.5 11 9.5 8.5
50.5 54.5 54 49.5 59.5
21.5 19 16.5 18 16.5
3 7.5 6.5 2 3.5
19 20 27 23 26
23 28.5 31 29.5 31
d: days after inoculation of culture.
© IRL Press Limited, Oxford, England
555
G.Chambat, N.Cartier and J.P.Joseleau
in the culture medium although in slightly different respective proportions (Table I). The overall composition of the cell walls showed an increase in the net content of neutral sugars. However, this increase did not correspond to a clear increase of the synthesis of a particular polysaccharide, as can be deduced from the sugar analysis. The extracellular medium on the other hand showed only discrete variations in the composition and nature of the released polysaccharides. As expected, the relative amount of uronic acids was much higher in the ECP. The mannose shows a particular trend in the culture medium where its proportion increases from 3 to 7 % and falls to 2 % in the stationary phase of the culture. This behavior was not observed in the cell wall analysis where the relative amount of mannose did not vary noticeably. Mannose-containing polysaccharides are not predominant in primary cell walls where only a few forms have been reported. Since it is easier to isolate and purify a polysaccharide from the extracellular medium than to extract it in pure form from the cell wall, the mannose-rich polysaccharide was studied mainly in the culture medium. After complexation of the pectin polymers with copper acetate (2), the supernatant was successively precipitated with ethanol and acetone. The last fraction was subjected to barium hydroxide purification. The resulting polysaccharide was shown to consist of a galactoglucomannan containing galactose, glucose and mannose in the molar ratio 1.0: 1.4: 1.2. Methylation analysis showed that more than two-thirds of the mannose was 4,6-linked and the rest 4-linked, whereas the glucose was mostly 4-linked and with only about 5 % of the residues carrying a substituent at 0-6. The 13C-NMR spectrum was clearly resolved with significant signals at 103.3 and 100.9 p.p.m. for {3(l-4) linked n-glucopyranosyl and n-mannopyranosyl residues respectively and at 105.3 and 99.8 p.p.m. for the substituting {3 and Q(u-galactopyranosyl residues (3). The main structural features were confirmed with the resonances of C-4 linked glucose and mannose at 79.8 and 79.5 p.p.m. and that of the substituted C-6 at 69.4 and 69.5 p.p.m. The same type of galactoglucomannan characteristic of primary wall could be isolated from an alkaline extract from the cell walls. Both methylation analysis and 13C-NMR showed that the extracellular galactoglucomannan was the counterpart of the wall polysaccharide. References I. Becker,G.E., Hui,P.A. and Albersheim,P. (1964) Plant Physiol., 39, 913-920. 2.Cartier,N., Chambat,G. and Joseleau,J.P. (1987) Carbohydr. Res., 168,275-283. 3. Akiyama,Y., Eda,S., Mori,M. and Kato,K. (1984) Agric. Bioi. Chem., 48, 403-408.
556
Extracellular polysaccharides from suspension-cultured cells of Rubus fruticosus. Structure of a galactoglucomannan G.Chambat, N.Cartier and J.P.Joseleau Centre de Recherches sur les Macromolecules Vegetales, B.P. 68, 38402 Saint-Martin d'Heres-Cedex, France Abstract. Carbohydrate analysis of the cell walls from suspension-cultured cells of Rubus fruticosus between day 10 and day 40 after inoculation revealed an overall increase of the net content of neutral sugars. However the composition of the extracellular polysaccharides showed only discrete variation. A galactoglucomannan containing galactose, glucose and mannose in the ratio 1.0: 1.4: 1.2 respectively, could be purified by barium hydroxide precipitation both in the extracellular medium and from the alkaline extract of the cell walls. Methylation analysis and 13C-NMR spectroscopy demonstrated that the extracellular form is the counterpart of the wall polysaccharide.
Introduction The extracellular medium of suspension-cultured cells is known to be rich in several types of water soluble polysaccharides. The extracellular polysaccharides (ECP) have been shown to have structures very much comparable to those of the cell wall polymers (CWP) (l). The mechanisms by which ECPs are released in the medium are not understood, neither is it known the possible roles that they may play in the cell culture. In the present work we analyzed the extracellular medium during Rubus fruticosus cell growth and compared it to the cell wall composition between days 10 and 40. We also report the isolation and structural determination of a typical galactoglucomannan from primary walls. Results Carbohydrate analysis of the cell walls at different ages and of the corresponding extracellular media revealed that all the sugars present in the cell wall are also found Table I. Changes in the composition of CWP and of ECP during the culture of Rifruticosus cells Uronic
Neutral
Neutral sugars (mol. %)
acids (wt %)
sugars (wt %)
Rhamnose
Fucose
dlO d 18 d 24 d 30 d 40
20.5 18.5 19.5 17.0 19.5
58.0 66.5 65.5 68.0 61.0
4 2.5 4 3.5 3
1.5 1.5 2 2.5 1.5
dlO d 18 d 24 d 30 d 40
28 30 27.5 27 27
50.5 45 43.5 49 35
6.5 4 3 4.5 4
5.5 4 3 3.5 4
Arabinose
CWP 19.5 19 16 20 12.5 ECP 21.5 17 13 19.5 IS
Xylose
Mannose
Galactose
Glucose
8.5 9.5 10.5 12 12.5
4 2.5 2.5 3 2.5
12 10.5 11 9.5 8.5
50.5 54.5 54 49.5 59.5
21.5 19 16.5 18 16.5
3 7.5 6.5 2 3.5
19 20 27 23 26
23 28.5 31 29.5 31
d: days after inoculation of culture.
© IRL Press Limited, Oxford, England
555
G.Chambat, N.Cartier and J.P.Joseleau
in the culture medium although in slightly different respective proportions (Table I). The overall composition of the cell walls showed an increase in the net content of neutral sugars. However, this increase did not correspond to a clear increase of the synthesis of a particular polysaccharide, as can be deduced from the sugar analysis. The extracellular medium on the other hand showed only discrete variations in the composition and nature of the released polysaccharides. As expected, the relative amount of uronic acids was much higher in the ECP. The mannose shows a particular trend in the culture medium where its proportion increases from 3 to 7 % and falls to 2 % in the stationary phase of the culture. This behavior was not observed in the cell wall analysis where the relative amount of mannose did not vary noticeably. Mannose-containing polysaccharides are not predominant in primary cell walls where only a few forms have been reported. Since it is easier to isolate and purify a polysaccharide from the extracellular medium than to extract it in pure form from the cell wall, the mannose-rich polysaccharide was studied mainly in the culture medium. After complexation of the pectin polymers with copper acetate (2), the supernatant was successively precipitated with ethanol and acetone. The last fraction was subjected to barium hydroxide purification. The resulting polysaccharide was shown to consist of a galactoglucomannan containing galactose, glucose and mannose in the molar ratio 1.0: 1.4: 1.2. Methylation analysis showed that more than two-thirds of the mannose was 4,6-linked and the rest 4-linked, whereas the glucose was mostly 4-linked and with only about 5 % of the residues carrying a substituent at 0-6. The 13C-NMR spectrum was clearly resolved with significant signals at 103.3 and 100.9 p.p.m. for {3(l-4) linked n-glucopyranosyl and n-mannopyranosyl residues respectively and at 105.3 and 99.8 p.p.m. for the substituting {3 and Q(u-galactopyranosyl residues (3). The main structural features were confirmed with the resonances of C-4 linked glucose and mannose at 79.8 and 79.5 p.p.m. and that of the substituted C-6 at 69.4 and 69.5 p.p.m. The same type of galactoglucomannan characteristic of primary wall could be isolated from an alkaline extract from the cell walls. Both methylation analysis and 13C-NMR showed that the extracellular galactoglucomannan was the counterpart of the wall polysaccharide. References I. Becker,G.E., Hui,P.A. and Albersheim,P. (1964) Plant Physiol., 39, 913-920. 2.Cartier,N., Chambat,G. and Joseleau,J.P. (1987) Carbohydr. Res., 168,275-283. 3. Akiyama,Y., Eda,S., Mori,M. and Kato,K. (1984) Agric. Bioi. Chem., 48, 403-408.
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