Chemistry and Physics of Lipids 14 (1975) 87-91 © NORTH-HOLLAND PUBLISHING COMPANY
LIPIDS IN PLANT TISSUE CULTURES V. EFFECT OF ENVIRONMENTAL CONDITIONS ON THE LIPIDS OF GL YCINE SOJA AND BRASSICA NAPUS CULTURES* S.S. RADWAN** and H.K. MANGOLD
Bundesanstalt fftr Fettforschung, 44 Mi~nster (lCestf.), Germany Received June 13, 1974, accepted August 12, 1974
The environmental cond itions prevailing during growth of plant tissue cultures affect the concentration of certain lipid classes and the fatty acid patterns of the total lipids. Irrespective of whether the cultures are grown under continuous illumination or in the dark, aerated cultures contain larger proportions of sterols, steryl esters, steryl glycosides and various sterylglycolipids and lower concentrations of squalene than non-aerated cultures. The lipids of the latter contain larger proportions of saturated very long-chain fatty acids than those of the former cultures. I..lntroduction Most of the lipid classes known to be present in higher plants have been reported to occur also in tissue cultures derived from these plants [ 1 - 6 ] . However, the factors influencing the lipid composition of plant tissue cultures have not been studied. The present communication describes the effect o f aeration and illumination on the growth, the lipid content and the lipid composition of suspension cultures derived from the roots of soybean (Glycine so~a) and rape (Brassica napus).
II. Experimental
A. Tissue cultures Seeds o f soybean (Glycine so~a) and rape (Brassica napus) were sterilized by soaking for 20 min in 0.25% aqueous mercury (II) chloride solution under reduced pressure. The seeds were thoroughly washed with sterile water, and allowed to germinate aseptically on wet filter paper in Petri dishes for three days. The seedlings * Part IV of this series is ref. 1. ** Recipient of an Alexander yon Humboldt-Stipendium, 1972-1974. Permanent address: Department of Botany, Faculty of Science, Ain Shams University, Cairo, Egypt.
88
S.S. Radwan, H.K. Mangold, Lipids in plant tissue cultures
were transferred onto solid RT 1 medium [7] in Petri dishes and, after three weeks, the callus cultures arising from the roots were inoculated in 50 ml aliquots of liquid medium RT0.1 [7], in Erlenmeyer flasks, in order to establish suspension cultures. These suspension cultures were allowed to develop at 26°C under the following conditions: (a) aeration under continuous illumination, (b) non-aeration under continuous illumination, (c) aeration in the dark, (d) non-aeration in the dark. Aeration was achieved by agitating the cultures on a shaker (80 strokes/rain). In some experiments, pure oxygen was supplied to study its effect on the lipids of nonaerated cultures. Fluorescent tubes were used for illumination. B. Lipid analysis
Tissues were harvested by filtration, and the lipids were extracted [8] and purified [9] following established procedures. Non-polar neutral lipids were fractioned by thin-layer chromatography on Silica Gel G with the solvent hexane-diethyl ether-acetic acid (90 : 10 : 1, v/v/v) [10]. Ionic and other polar lipids were resolved by thin-layer chromatography on Silica Gel H with the solvents c h l o r o f o r m - m e t h a n o l - a c e t i c acid (80 : 25 : 1, v/v/v)_[8] and c h l o r o f o r m - m e t h a n o l - w a t e r (65 : 25 : 4, v/v/v) [11 ]. Lipid fractions were detected by charring after spraying the plates with 50% aqueous sulfuric acid solution. The various lipid classes were identified by comparison of their migration rates with those of authentic samples and by their color reactions with various spray reagents [12, 13]. For quantitative analyses of the various lipid classes, known amounts of total lipids were fractioned by chromatography on layers of Silica Gel H, 0.5 mm thick, using the solvents specified above. The lipids were eluted with water-saturated diethyl ether, chloroform methanol diethyl ether (1 : 1 : 1, v/v/v) [14] or chloroform methanol-water (3 : 5 : 2, v/v/v) [15] and weighed. Aliquots of the pure lipid fractions isolated were subjected to methanolysis and the resulting methyl esters were purified by thin-layer chromatography [ 16]. Gas chromatographic analyses of the methyl esters were carried out on a Hewlett-Packard Model 5750 G instrument equipped with a flame ionization detector using a column, 6 ft × g , packed with 15% DEGS on Anakrom D, 100/120 mesh, at 176°C.
Ill. Results and discussion
We have found that the growth of aerated suspension cultures of soybean as well as of rape was better than that of non-aerated cultures. Table 1 shows that this holds true for both cultures grown under continuous illumination and those grown in the dark.
1.19 1.48
1 2
1.29 1.31
63.9 58.0
1.16 1.22
(%)
(%)
74.8 64.3
Alicyclic lipidsb GIa
Alicyclic lipid sb Gla
62.3 52.0
(%) 1.20 1.22
Alicyclic lipidsb Gla
50.3
(%) 4.45 1.42
Alicyclic lipidsb GIa
50.6 53.5
(%) 3.69 1.37
Alicyclic lipidsb Gla
in dark
50.0 61.5
(%) 2.28 1.16
Alicyclic lipidsb GIa
aGl: Growth index values of 1 week old cultures
in dark
34.0 25.3
(%) 2.21 1.23
Alicyclic lipidsb Gla
in light
Non-aerated cultures
final tissue weight after 1 week of growth weight of inoculum weight of 2 weeks old tissues Growth index values of 2 weeks old cultures = weight of 1 week old tissues bAlicyclic lipids: Sterols, steryl esters and esterified steryl glycosides. Results are expressed in percentage of the total lipids.
Gla
Age (weeks)
in light
in light
in light
in dark
Aerated cultures
Non-aerated cultures
Aerated cultures
in dark
Rape
Soybean
Table 1 Sterols, steryl esters and esterified steryl glycosides of soybean and rape suspension cultures.
36.2 27.5
(%)
Alicyclic lipidsb
O~
r~
e~
E'-
5"
t',
90
S.S. Radwan, H.K. Mangold, Lipids in plant tissue cultures
The lipid content of both soybean and rape cultures was between 7 and 8% of the dry weight, regardless of the environmental conditions and irrespective of the age of the cultures. Regardless of the environmental conditions, the cultures contained large proportions of sterols, steryl esters and esterified steryl glycosides. Squalene was found in appreciable amounts, especially in non-aerated cultures. Both in cultures grown under continuous illumination and in those grown in the dark, phospholipids and triglycerides occurred in relatively small proportions whereas monogalactosyl diglycerides and digalactosyl diglycerides as well as sulfoquinovosyl diglycerides were present only in traces, if at all. It is evident from table 1 that in both soybean and rape cultures aeration stimulated the synthesis of alicyclic lipids regardless of the light conditions and the age of the cultures. This increase in alicyclic lipids was accompanied by a decrease in squalene. The results presented in table 2 show that the synthesis of alicyclic lipids from squalene in non-aerated cultures can be enhanced by supplying oxygen. This result is not surprising in view of the fact that the cyclization of squalene to sterols in plants is known to require molecular oxygen [17]. We have found that the levels of aliphatic lipids were not affected by aeration but the constituent fatty acids o f lipids extracted from non-aerated cultures contained rather large proportions of saturated very long-chain fatty acids. Such very long-chain fatty acids were found as constituents of lipids in a variety of other plant tissue cultures as well [18]. The chain elongation of fatty acids in plants requires reduced pyridine nucleotides [19], which are known to accumulate in the absence of oxygen. This may indicate that elongation occurs preferentially in tissues growing under low oxygen supply. The results of the present communication support this view. Table 2 Effect of oxygen on the lipids of non-aerated soybean and rape suspension cultures. Soybean
non-aerated a
%
non-aerated plus 02 b %
Squalene
25.0
13.0
26.4
6.8
Alicyclic lipids
52.0
60.0
25.3
52.0
Lipids
non-aerated a
Rape
%
Data are expressed in percentage of the total lipids. a The cultures were grown without aeration for 2 weeks in light at 26°C. b As in (a), then supplied with 02 for 24 hours in light at 26°C.
non-aerated plus 02 b %
S.S. Radwan, H.K. Mangold, Lipids in plant tissue cultures
91
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [ 10] [11] [12] [ 131 [14] [15] [16] [17] [181 [191
S.S. Radwan, F. Spener, H.K. Mangold and E.J. Staba, Chem. Phys. Lipids W.H. Davies, E.I. Mercer and T.W. Goodwin, Phytochemistry 4 (1965) 741 E.C. Grob and W. Eichenberger, FEBS Letters 5 (1969) 335 B.S. Shin, H.K. Marigold and E.J. Staba, in: Colloques Internationaux C.N.R.S. No. 193 Les Cultures de Tissus de Plantes. Paris (1971) 51 N.H. Tattrie and I.A. Veliky, Can. J. Botany 51 (1973) 513 F. Spener, E.J. Staba and H.K. Mangold, Chem. Phys. Lipids 12 (1974) 344 T. Murashige and F. Skoog, Physiol. Plantarum 15 (1962) 473 B.W. Nichols, in: New biochemical separations, ed. by A.T. James and L.J. Morris. Van Nostrand, London, New York, Toronto (1964) 321 J. Folch, M. Lees and G.H. Sloane Stanley, J. Biol. Chem. 226 (1957) 497 H.K. Mangoid and D.C. Malins, J. Am. Oil Chemists' Soc. 37 (1960) 383 H. Wagner, L. H~Srhammer and P. Wolff, Biochem. Z, 334 (1961) 175 J.C. Dittmer and R.L. Lester, J. Lipid Res. 5 (1964) 126 A.N. Siakotos and G. Rouser, J. Am. Oil Chemists' Soc. 42 (1965) 913 M. Kates, Techniques of lipidology, North Holland, Amsterdam (1972) 444 K. Slotta, Monatsh. Chem. 97 (1966) 1723 A.M. Chalvardjian, Biochem. J. 90 (1964) 518 T.T. Tchen and K. Bloch, J. Am. Chem. Soc. 78 (1956) 1516 S.S. Radwan, H.K. Marigold and F. Spener, Chem. Phys. Lipids 13 (1974) 103 D.E. Vance, O. Mitsuhashi and K. Bloch, J. Biol. Chem. 248 (1973) 2303
LIPIDS IN PLANT TISSUE CULTURES V. EFFECT OF ENVIRONMENTAL CONDITIONS ON THE LIPIDS OF GL YCINE SOJA AND BRASSICA NAPUS CULTURES* S.S. RADWAN** and H.K. MANGOLD
Bundesanstalt fftr Fettforschung, 44 Mi~nster (lCestf.), Germany Received June 13, 1974, accepted August 12, 1974
The environmental cond itions prevailing during growth of plant tissue cultures affect the concentration of certain lipid classes and the fatty acid patterns of the total lipids. Irrespective of whether the cultures are grown under continuous illumination or in the dark, aerated cultures contain larger proportions of sterols, steryl esters, steryl glycosides and various sterylglycolipids and lower concentrations of squalene than non-aerated cultures. The lipids of the latter contain larger proportions of saturated very long-chain fatty acids than those of the former cultures. I..lntroduction Most of the lipid classes known to be present in higher plants have been reported to occur also in tissue cultures derived from these plants [ 1 - 6 ] . However, the factors influencing the lipid composition of plant tissue cultures have not been studied. The present communication describes the effect o f aeration and illumination on the growth, the lipid content and the lipid composition of suspension cultures derived from the roots of soybean (Glycine so~a) and rape (Brassica napus).
II. Experimental
A. Tissue cultures Seeds o f soybean (Glycine so~a) and rape (Brassica napus) were sterilized by soaking for 20 min in 0.25% aqueous mercury (II) chloride solution under reduced pressure. The seeds were thoroughly washed with sterile water, and allowed to germinate aseptically on wet filter paper in Petri dishes for three days. The seedlings * Part IV of this series is ref. 1. ** Recipient of an Alexander yon Humboldt-Stipendium, 1972-1974. Permanent address: Department of Botany, Faculty of Science, Ain Shams University, Cairo, Egypt.
88
S.S. Radwan, H.K. Mangold, Lipids in plant tissue cultures
were transferred onto solid RT 1 medium [7] in Petri dishes and, after three weeks, the callus cultures arising from the roots were inoculated in 50 ml aliquots of liquid medium RT0.1 [7], in Erlenmeyer flasks, in order to establish suspension cultures. These suspension cultures were allowed to develop at 26°C under the following conditions: (a) aeration under continuous illumination, (b) non-aeration under continuous illumination, (c) aeration in the dark, (d) non-aeration in the dark. Aeration was achieved by agitating the cultures on a shaker (80 strokes/rain). In some experiments, pure oxygen was supplied to study its effect on the lipids of nonaerated cultures. Fluorescent tubes were used for illumination. B. Lipid analysis
Tissues were harvested by filtration, and the lipids were extracted [8] and purified [9] following established procedures. Non-polar neutral lipids were fractioned by thin-layer chromatography on Silica Gel G with the solvent hexane-diethyl ether-acetic acid (90 : 10 : 1, v/v/v) [10]. Ionic and other polar lipids were resolved by thin-layer chromatography on Silica Gel H with the solvents c h l o r o f o r m - m e t h a n o l - a c e t i c acid (80 : 25 : 1, v/v/v)_[8] and c h l o r o f o r m - m e t h a n o l - w a t e r (65 : 25 : 4, v/v/v) [11 ]. Lipid fractions were detected by charring after spraying the plates with 50% aqueous sulfuric acid solution. The various lipid classes were identified by comparison of their migration rates with those of authentic samples and by their color reactions with various spray reagents [12, 13]. For quantitative analyses of the various lipid classes, known amounts of total lipids were fractioned by chromatography on layers of Silica Gel H, 0.5 mm thick, using the solvents specified above. The lipids were eluted with water-saturated diethyl ether, chloroform methanol diethyl ether (1 : 1 : 1, v/v/v) [14] or chloroform methanol-water (3 : 5 : 2, v/v/v) [15] and weighed. Aliquots of the pure lipid fractions isolated were subjected to methanolysis and the resulting methyl esters were purified by thin-layer chromatography [ 16]. Gas chromatographic analyses of the methyl esters were carried out on a Hewlett-Packard Model 5750 G instrument equipped with a flame ionization detector using a column, 6 ft × g , packed with 15% DEGS on Anakrom D, 100/120 mesh, at 176°C.
Ill. Results and discussion
We have found that the growth of aerated suspension cultures of soybean as well as of rape was better than that of non-aerated cultures. Table 1 shows that this holds true for both cultures grown under continuous illumination and those grown in the dark.
1.19 1.48
1 2
1.29 1.31
63.9 58.0
1.16 1.22
(%)
(%)
74.8 64.3
Alicyclic lipidsb GIa
Alicyclic lipid sb Gla
62.3 52.0
(%) 1.20 1.22
Alicyclic lipidsb Gla
50.3
(%) 4.45 1.42
Alicyclic lipidsb GIa
50.6 53.5
(%) 3.69 1.37
Alicyclic lipidsb Gla
in dark
50.0 61.5
(%) 2.28 1.16
Alicyclic lipidsb GIa
aGl: Growth index values of 1 week old cultures
in dark
34.0 25.3
(%) 2.21 1.23
Alicyclic lipidsb Gla
in light
Non-aerated cultures
final tissue weight after 1 week of growth weight of inoculum weight of 2 weeks old tissues Growth index values of 2 weeks old cultures = weight of 1 week old tissues bAlicyclic lipids: Sterols, steryl esters and esterified steryl glycosides. Results are expressed in percentage of the total lipids.
Gla
Age (weeks)
in light
in light
in light
in dark
Aerated cultures
Non-aerated cultures
Aerated cultures
in dark
Rape
Soybean
Table 1 Sterols, steryl esters and esterified steryl glycosides of soybean and rape suspension cultures.
36.2 27.5
(%)
Alicyclic lipidsb
O~
r~
e~
E'-
5"
t',
90
S.S. Radwan, H.K. Mangold, Lipids in plant tissue cultures
The lipid content of both soybean and rape cultures was between 7 and 8% of the dry weight, regardless of the environmental conditions and irrespective of the age of the cultures. Regardless of the environmental conditions, the cultures contained large proportions of sterols, steryl esters and esterified steryl glycosides. Squalene was found in appreciable amounts, especially in non-aerated cultures. Both in cultures grown under continuous illumination and in those grown in the dark, phospholipids and triglycerides occurred in relatively small proportions whereas monogalactosyl diglycerides and digalactosyl diglycerides as well as sulfoquinovosyl diglycerides were present only in traces, if at all. It is evident from table 1 that in both soybean and rape cultures aeration stimulated the synthesis of alicyclic lipids regardless of the light conditions and the age of the cultures. This increase in alicyclic lipids was accompanied by a decrease in squalene. The results presented in table 2 show that the synthesis of alicyclic lipids from squalene in non-aerated cultures can be enhanced by supplying oxygen. This result is not surprising in view of the fact that the cyclization of squalene to sterols in plants is known to require molecular oxygen [17]. We have found that the levels of aliphatic lipids were not affected by aeration but the constituent fatty acids o f lipids extracted from non-aerated cultures contained rather large proportions of saturated very long-chain fatty acids. Such very long-chain fatty acids were found as constituents of lipids in a variety of other plant tissue cultures as well [18]. The chain elongation of fatty acids in plants requires reduced pyridine nucleotides [19], which are known to accumulate in the absence of oxygen. This may indicate that elongation occurs preferentially in tissues growing under low oxygen supply. The results of the present communication support this view. Table 2 Effect of oxygen on the lipids of non-aerated soybean and rape suspension cultures. Soybean
non-aerated a
%
non-aerated plus 02 b %
Squalene
25.0
13.0
26.4
6.8
Alicyclic lipids
52.0
60.0
25.3
52.0
Lipids
non-aerated a
Rape
%
Data are expressed in percentage of the total lipids. a The cultures were grown without aeration for 2 weeks in light at 26°C. b As in (a), then supplied with 02 for 24 hours in light at 26°C.
non-aerated plus 02 b %
S.S. Radwan, H.K. Mangold, Lipids in plant tissue cultures
91
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [ 10] [11] [12] [ 131 [14] [15] [16] [17] [181 [191
S.S. Radwan, F. Spener, H.K. Mangold and E.J. Staba, Chem. Phys. Lipids W.H. Davies, E.I. Mercer and T.W. Goodwin, Phytochemistry 4 (1965) 741 E.C. Grob and W. Eichenberger, FEBS Letters 5 (1969) 335 B.S. Shin, H.K. Marigold and E.J. Staba, in: Colloques Internationaux C.N.R.S. No. 193 Les Cultures de Tissus de Plantes. Paris (1971) 51 N.H. Tattrie and I.A. Veliky, Can. J. Botany 51 (1973) 513 F. Spener, E.J. Staba and H.K. Mangold, Chem. Phys. Lipids 12 (1974) 344 T. Murashige and F. Skoog, Physiol. Plantarum 15 (1962) 473 B.W. Nichols, in: New biochemical separations, ed. by A.T. James and L.J. Morris. Van Nostrand, London, New York, Toronto (1964) 321 J. Folch, M. Lees and G.H. Sloane Stanley, J. Biol. Chem. 226 (1957) 497 H.K. Mangoid and D.C. Malins, J. Am. Oil Chemists' Soc. 37 (1960) 383 H. Wagner, L. H~Srhammer and P. Wolff, Biochem. Z, 334 (1961) 175 J.C. Dittmer and R.L. Lester, J. Lipid Res. 5 (1964) 126 A.N. Siakotos and G. Rouser, J. Am. Oil Chemists' Soc. 42 (1965) 913 M. Kates, Techniques of lipidology, North Holland, Amsterdam (1972) 444 K. Slotta, Monatsh. Chem. 97 (1966) 1723 A.M. Chalvardjian, Biochem. J. 90 (1964) 518 T.T. Tchen and K. Bloch, J. Am. Chem. Soc. 78 (1956) 1516 S.S. Radwan, H.K. Marigold and F. Spener, Chem. Phys. Lipids 13 (1974) 103 D.E. Vance, O. Mitsuhashi and K. Bloch, J. Biol. Chem. 248 (1973) 2303
