Planta
Planta 134, 115-117 (1977)
9 by Springer-Verlag 1977
Sterols of Male and Female Flowers of Cucumis sativus B.A. Knights and Aileen R. Smith Department of Botany, University of Glasgow, Glasgow G12 8QQ, U.K.
Abstract. Sterols of male and female flowers of Cucumis sativus L. were similar in composition. The principal compound was 24~-ethyl-5~-cholesta-7,22dien-3fi-ol. Five other 5c~-AT-sterols were detected: 24~-methyl-7-ene, 24~-ethyl-7-ene, 24-ethyl-7,24(28)Zdiene, 24~-ethyl-7,25-diene and 24~-ethyl-7,22,25-triene. Small amounts of AS-sterol (cholesterol, 244methylcholesterol and 24~-ethylcholesterol) were detected. The possible significance of these sterols is discussed.
Key words: Female - Flowers - Male - Meiosis Sterols.
Introduction
It has been known for some time that the fungus Phytophthora cactorum is unable to synthesise sterols
and that an external source of them is required for asexual and sexual reproduction to occur (Hendrix, 1970). Elliott et al. (1966), Elliott (1972) have considered the structural features of sterols which induce sexual reproduction (formation of antheridia and oogonia followed by fusion to produce oospores). They find that a number of A 5_ or A7-C27/C29 sterols served the purpose and cholesterol is the commonly employed reference compound. Ring B-saturated sterols promoted formation of oogonia or antheridia but did not promote oospore formation. Elliott (1968) showed that a sparing action of cholestanoI upon cholesterol existed at low concentrations but that high levels of this saturated sterol were inhibitory of oospore production, implying a competitive binding effect at one of at least two sites of action of sterols. Abbreviations," GLC = gas-liquid chromatography ; GC-MS =combined gas chromatography-mass spectrometry
Recently, Elliott and Sansome (1977) have shown that P. cactorum when grown on a cholestanol supplemented medium, produced oogonia which aborted and that in these oogonia meiosis had been inhibited at or before early prophase. Knights and Elliott (1976) have shown that A7 sterols are metabolised to A5- by P. cactorum. In addition, comparison of the relative activities of A 5_ and A~-sterols over a wide range of concentrations (Elliott, personal communication) has led to the belief that a specific requirement for A S-sterol exists in P. cactorum for sexual reproduction to occur. It is considered possible that this requirement may be associated with the process of meiosis in P. cactorurn. This raises the interesting possibility of an important rote for sterols in the nucleus at meiosis generally and of the added possibility that a specific structural requirement for a A S-sterol exists as general phenomenon. To observe this further requires another class of organism which, like P. cactorum, has no endogenous sterol or, alternatively, an organism in which little or no A5-sterol exists. Representative of the former class are insects, where dietary sterols are required for the production of the moulting hormone ecdysterone. In addition it has been shown that cholestanol can spare the effects of cholesterol in insects (Clayton and Bloch, 1963). Nes (1974) has claimed that members of the Cucurbitaceae do not synthesise A S-sterol. This statement is refuted by the finding of (24S)24-ethyl-5,25-cholestadien-3/%ol in Momordica charantia by Sucrow (1966) and also is not compatible with the belief that cholesterol is a component of sterols in chloroplasts (Knights, 1971). However, species of Cucumis very largely contain AT-sterols, only small amounts of A~-sterols having been isolated from seed and seedlings (Sucrow and Reimerdes, 1968; Sucrow and Radfichel, 1971; Kintia and Wojciechowski, 1974). Since this genus is normally monoecious it has the additional advantage that male
116
B.A. Knights and A.R. Smith: Sterols of Flowers
and female flowers occur separately on the same plant. It was decided to examine the sterols of male (staminate) and female (carpellate) flowers o f C. sativus in order to see whether A S-sterols are present in either or both cases; if so this species could serve to test further the role of AS-sterols in meiosis in either male or female flowers in plants.
Cucumis sativus o" TMS 0V-17 3
2
1
1
Materials and Methods
a
Cucumber variety Telegraph was grown in a greenhouse border of the departmental experimental garden. Flowers were stored in methanol at 2~ until used for extraction. Sterols were isolated and analysed using previously described methods (Knights and Smith, 1976).
2'5
20
15 min
Cucumis sativus
9, B
Results
Sterols from male flowers and from female flowers with and without ovaries were analysed by GLC and GC-MS. The main compounds so determined were 24~-ethyl-5e-cholesta-7,22-dien-3fl-ol (1), 24~-ethyl5~-cholest-7-en-3fl-ol (2), 24~-ethyl-5c~-cholest-7,25dien-3/%ol (3) and 28Z-24-ethylidene-5c~-cholest-7-en3/~-ol (AT-avenasterol) (4). These data agree with earlier reports for this species (Sucrow and Reimerdes, 1968; Kintia and Wojciechowski, 1974). In addition, mass spectral data for small amounts of 24~-ethylcholesterol (m/e 486, 396, 381, 357) and 24~-methylcholesterol (m/e 472, 382, 343) were obtained in the sterol fraction from male flowers. The same two A 2_ compounds plus cholesterol were detected in female flowers containing ovaries. MS evidence was not obtained from female flowers without ovaries but
2
I
l
25
20
15 min
Cucurnis sativus
~A
1
2
3
1
C2Hs
C2H5
l
25
20
15 rnin
Fig. 1. GLC. traces o f TMS derivatives of sterols from carpellate (~) and staminate (d') flowers from C. sativus. Numbered arrows indicate positions at which mass spectral scans were taken for
(1)
(2) .C2Hs
(3)
(4)
main peaks. 1: compound (1), 2: compounds (2) and (3), 3 and 4: compound (4) plus 24-methylenecycloartanol. (a) and (b) denote the positionsfor the A5-sterolscholesteroland 24-methylcholesterol respectively. 24-ethylcholesterolchromatographs with peak 1 and could be detected in mass spectra
GLC data suggested the possibility of cholesterol and 24-methylcholesterol being present. A trace of a presumed C29-7,22,25-trienol was also detected in all samples. Representative GLC traces are shown in Figure 1.
B.A. Knights and A.R. Smith: Sterols of Flowers
117
Discussion
References
In an earlier study of sterols of the leaves and isolated chloroplasts of Cucumis sativus (Knights et al., unpublished) we observed the principal A 7-sterols to be the same as were identified in this work. Cholesterol could be detected in chloroplasts as was expected from earlier findings (Knights, 197t) and small amounts of other A 5-sterols were also present in the extracts. Kintia and Wojciechowski (1974) record the presence of A S-mono and diunsaturated sterols in the glycoside fraction of C. sativus seedlings (10 days old) but they do not mention such compounds as occurring in the free (i.e. not derivatised) sterol fraction. In our experiments only free sterols were analysed and although they form only minor components, A 5-sterols are certainly present in both male and female flowers of C. sativus. The results of Kintia and Wojciechowski (1974) imply that sterol metabolism in this species is not a simple equilibrium process; that is, the free esterified and glucosylated fractions do not have identical compositions. Such findings are fairly common in plant species (Knights, 1973) and this is especially true of the sterol fraction from nuclei and nuclear enriched subcellular particulate fractions (Kemp and Mercer, 1968; Brandt and Benveniste, 1972). In these cases the relative occurrence of cholesterol amongst free sterols was highest in those fractions enriched in nuclei. Although we have no data to suggest that A 5sterols occur at any one location within cells of C. sativus, their presence as free sterols in flowers is sufficient to encourage us in the belief that it is worth making a study of the specific location, biosynthesis and metabolism of this group of compounds in this species, especially in relation to any possible involvement in meiosis.
Brandt, R.D., Benveniste, P. : Isolation and identification of sterols from subcellular fractions of bean leaves. Biochim. Biophys. Acta 282, 85-92 (1972) Clayton, R.B., Bloch, K,: Sterol utilization in the hide Beetle, Dermestes vulpinus. J. biol. Chem. 238, 586 (1963) Elliott, C.G., Hendrie, M.R., Knights, B.A.: The sterol requirement of Phytophthora eactorum. J. gem Microbiol. 42, 425~-35 (1966) Elliott, C.G. : Competition and synergism between cholesterol and cholestanoI in oospore formation in Phythophthora caetorum. J. gem Microbiol. 51, 137 143 (1968) Elliott, C.G.: Sterols and the production of oospores by Phytophthora cactorum. J. gen. Microbiol. 72, 321-327 (1972) Elliott, C.G., Sansome, E.: Sterols and meiosis in Phytophthora. J. gen. Microbiol. 98, 141 145 (1977) Hendrix, J.W. : Sterols in Growth and Reproduction of fungi. Ann. Rev. Phytopathol. 8, 11t 130 (1970) Kemp, R.J., Mercer, E.I.: Studies on the sterols and sterol esters of the intracellular organelles of maize shoots. Biochem. J. 110, 119 (1968) Kintia, P.K., Wojciechowski, Z.A.: Free and bound sterol in seedlings of Cucumis sativus. Phytochem. 13, 2235 (1974) Knights, B.A. : Sterol metabolism in plants. Chem. Brit. 9, 106-111 (1973) Knights, B.A., Elliott, C.G.: Metabolism of A 7- and AS'7-sterols by Phytophthora cactorum. Biochim. Biophys. Acta. 441, 341346 (1976) Knights, B.A., Smith, A.R. : Sterols of male and female compound inflorescences of Zea mays. Planta 33, 89-93 (1976) Nes, W.R. : Role of sterols in membranes. Lipids 9, 596-612 (1974) Sucrow, W.: Inhaltsstoffe von Momordica charantia L. A s~zsStigmastadienol-(3/~) und sein/~-D-glucosid. Chem. Ber. 99, 2765 (1966) Sucrow, W., Reimerdes, A.: AT-sterols from Cucmbitaceae. Z. Naturforsch. 23, 5~54 (1968) Sucrow, W., Radtichel, B. : Biosynthesis of c~-spinasterol in pumpkin. Tetrahedron 27, 4097 (1971)
Received 3 September; accepted 19 November 1976
Planta 134, 115-117 (1977)
9 by Springer-Verlag 1977
Sterols of Male and Female Flowers of Cucumis sativus B.A. Knights and Aileen R. Smith Department of Botany, University of Glasgow, Glasgow G12 8QQ, U.K.
Abstract. Sterols of male and female flowers of Cucumis sativus L. were similar in composition. The principal compound was 24~-ethyl-5~-cholesta-7,22dien-3fi-ol. Five other 5c~-AT-sterols were detected: 24~-methyl-7-ene, 24~-ethyl-7-ene, 24-ethyl-7,24(28)Zdiene, 24~-ethyl-7,25-diene and 24~-ethyl-7,22,25-triene. Small amounts of AS-sterol (cholesterol, 244methylcholesterol and 24~-ethylcholesterol) were detected. The possible significance of these sterols is discussed.
Key words: Female - Flowers - Male - Meiosis Sterols.
Introduction
It has been known for some time that the fungus Phytophthora cactorum is unable to synthesise sterols
and that an external source of them is required for asexual and sexual reproduction to occur (Hendrix, 1970). Elliott et al. (1966), Elliott (1972) have considered the structural features of sterols which induce sexual reproduction (formation of antheridia and oogonia followed by fusion to produce oospores). They find that a number of A 5_ or A7-C27/C29 sterols served the purpose and cholesterol is the commonly employed reference compound. Ring B-saturated sterols promoted formation of oogonia or antheridia but did not promote oospore formation. Elliott (1968) showed that a sparing action of cholestanoI upon cholesterol existed at low concentrations but that high levels of this saturated sterol were inhibitory of oospore production, implying a competitive binding effect at one of at least two sites of action of sterols. Abbreviations," GLC = gas-liquid chromatography ; GC-MS =combined gas chromatography-mass spectrometry
Recently, Elliott and Sansome (1977) have shown that P. cactorum when grown on a cholestanol supplemented medium, produced oogonia which aborted and that in these oogonia meiosis had been inhibited at or before early prophase. Knights and Elliott (1976) have shown that A7 sterols are metabolised to A5- by P. cactorum. In addition, comparison of the relative activities of A 5_ and A~-sterols over a wide range of concentrations (Elliott, personal communication) has led to the belief that a specific requirement for A S-sterol exists in P. cactorum for sexual reproduction to occur. It is considered possible that this requirement may be associated with the process of meiosis in P. cactorurn. This raises the interesting possibility of an important rote for sterols in the nucleus at meiosis generally and of the added possibility that a specific structural requirement for a A S-sterol exists as general phenomenon. To observe this further requires another class of organism which, like P. cactorum, has no endogenous sterol or, alternatively, an organism in which little or no A5-sterol exists. Representative of the former class are insects, where dietary sterols are required for the production of the moulting hormone ecdysterone. In addition it has been shown that cholestanol can spare the effects of cholesterol in insects (Clayton and Bloch, 1963). Nes (1974) has claimed that members of the Cucurbitaceae do not synthesise A S-sterol. This statement is refuted by the finding of (24S)24-ethyl-5,25-cholestadien-3/%ol in Momordica charantia by Sucrow (1966) and also is not compatible with the belief that cholesterol is a component of sterols in chloroplasts (Knights, 1971). However, species of Cucumis very largely contain AT-sterols, only small amounts of A~-sterols having been isolated from seed and seedlings (Sucrow and Reimerdes, 1968; Sucrow and Radfichel, 1971; Kintia and Wojciechowski, 1974). Since this genus is normally monoecious it has the additional advantage that male
116
B.A. Knights and A.R. Smith: Sterols of Flowers
and female flowers occur separately on the same plant. It was decided to examine the sterols of male (staminate) and female (carpellate) flowers o f C. sativus in order to see whether A S-sterols are present in either or both cases; if so this species could serve to test further the role of AS-sterols in meiosis in either male or female flowers in plants.
Cucumis sativus o" TMS 0V-17 3
2
1
1
Materials and Methods
a
Cucumber variety Telegraph was grown in a greenhouse border of the departmental experimental garden. Flowers were stored in methanol at 2~ until used for extraction. Sterols were isolated and analysed using previously described methods (Knights and Smith, 1976).
2'5
20
15 min
Cucumis sativus
9, B
Results
Sterols from male flowers and from female flowers with and without ovaries were analysed by GLC and GC-MS. The main compounds so determined were 24~-ethyl-5e-cholesta-7,22-dien-3fl-ol (1), 24~-ethyl5~-cholest-7-en-3fl-ol (2), 24~-ethyl-5c~-cholest-7,25dien-3/%ol (3) and 28Z-24-ethylidene-5c~-cholest-7-en3/~-ol (AT-avenasterol) (4). These data agree with earlier reports for this species (Sucrow and Reimerdes, 1968; Kintia and Wojciechowski, 1974). In addition, mass spectral data for small amounts of 24~-ethylcholesterol (m/e 486, 396, 381, 357) and 24~-methylcholesterol (m/e 472, 382, 343) were obtained in the sterol fraction from male flowers. The same two A 2_ compounds plus cholesterol were detected in female flowers containing ovaries. MS evidence was not obtained from female flowers without ovaries but
2
I
l
25
20
15 min
Cucurnis sativus
~A
1
2
3
1
C2Hs
C2H5
l
25
20
15 rnin
Fig. 1. GLC. traces o f TMS derivatives of sterols from carpellate (~) and staminate (d') flowers from C. sativus. Numbered arrows indicate positions at which mass spectral scans were taken for
(1)
(2) .C2Hs
(3)
(4)
main peaks. 1: compound (1), 2: compounds (2) and (3), 3 and 4: compound (4) plus 24-methylenecycloartanol. (a) and (b) denote the positionsfor the A5-sterolscholesteroland 24-methylcholesterol respectively. 24-ethylcholesterolchromatographs with peak 1 and could be detected in mass spectra
GLC data suggested the possibility of cholesterol and 24-methylcholesterol being present. A trace of a presumed C29-7,22,25-trienol was also detected in all samples. Representative GLC traces are shown in Figure 1.
B.A. Knights and A.R. Smith: Sterols of Flowers
117
Discussion
References
In an earlier study of sterols of the leaves and isolated chloroplasts of Cucumis sativus (Knights et al., unpublished) we observed the principal A 7-sterols to be the same as were identified in this work. Cholesterol could be detected in chloroplasts as was expected from earlier findings (Knights, 197t) and small amounts of other A 5-sterols were also present in the extracts. Kintia and Wojciechowski (1974) record the presence of A S-mono and diunsaturated sterols in the glycoside fraction of C. sativus seedlings (10 days old) but they do not mention such compounds as occurring in the free (i.e. not derivatised) sterol fraction. In our experiments only free sterols were analysed and although they form only minor components, A 5-sterols are certainly present in both male and female flowers of C. sativus. The results of Kintia and Wojciechowski (1974) imply that sterol metabolism in this species is not a simple equilibrium process; that is, the free esterified and glucosylated fractions do not have identical compositions. Such findings are fairly common in plant species (Knights, 1973) and this is especially true of the sterol fraction from nuclei and nuclear enriched subcellular particulate fractions (Kemp and Mercer, 1968; Brandt and Benveniste, 1972). In these cases the relative occurrence of cholesterol amongst free sterols was highest in those fractions enriched in nuclei. Although we have no data to suggest that A 5sterols occur at any one location within cells of C. sativus, their presence as free sterols in flowers is sufficient to encourage us in the belief that it is worth making a study of the specific location, biosynthesis and metabolism of this group of compounds in this species, especially in relation to any possible involvement in meiosis.
Brandt, R.D., Benveniste, P. : Isolation and identification of sterols from subcellular fractions of bean leaves. Biochim. Biophys. Acta 282, 85-92 (1972) Clayton, R.B., Bloch, K,: Sterol utilization in the hide Beetle, Dermestes vulpinus. J. biol. Chem. 238, 586 (1963) Elliott, C.G., Hendrie, M.R., Knights, B.A.: The sterol requirement of Phytophthora eactorum. J. gem Microbiol. 42, 425~-35 (1966) Elliott, C.G. : Competition and synergism between cholesterol and cholestanoI in oospore formation in Phythophthora caetorum. J. gem Microbiol. 51, 137 143 (1968) Elliott, C.G.: Sterols and the production of oospores by Phytophthora cactorum. J. gen. Microbiol. 72, 321-327 (1972) Elliott, C.G., Sansome, E.: Sterols and meiosis in Phytophthora. J. gen. Microbiol. 98, 141 145 (1977) Hendrix, J.W. : Sterols in Growth and Reproduction of fungi. Ann. Rev. Phytopathol. 8, 11t 130 (1970) Kemp, R.J., Mercer, E.I.: Studies on the sterols and sterol esters of the intracellular organelles of maize shoots. Biochem. J. 110, 119 (1968) Kintia, P.K., Wojciechowski, Z.A.: Free and bound sterol in seedlings of Cucumis sativus. Phytochem. 13, 2235 (1974) Knights, B.A. : Sterol metabolism in plants. Chem. Brit. 9, 106-111 (1973) Knights, B.A., Elliott, C.G.: Metabolism of A 7- and AS'7-sterols by Phytophthora cactorum. Biochim. Biophys. Acta. 441, 341346 (1976) Knights, B.A., Smith, A.R. : Sterols of male and female compound inflorescences of Zea mays. Planta 33, 89-93 (1976) Nes, W.R. : Role of sterols in membranes. Lipids 9, 596-612 (1974) Sucrow, W.: Inhaltsstoffe von Momordica charantia L. A s~zsStigmastadienol-(3/~) und sein/~-D-glucosid. Chem. Ber. 99, 2765 (1966) Sucrow, W., Reimerdes, A.: AT-sterols from Cucmbitaceae. Z. Naturforsch. 23, 5~54 (1968) Sucrow, W., Radtichel, B. : Biosynthesis of c~-spinasterol in pumpkin. Tetrahedron 27, 4097 (1971)
Received 3 September; accepted 19 November 1976
