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Estrogens in the short-day plants Perilla ocimoides and Chenopodium rubrum grown under inductive and noninductive light conditions
Department of Plant Physiology, Institute of Biology, Copernicus University, Toruil, Poland.
Estrogens in the Short-day Plants Perilla ocimoides and Chenopodium rubrum Grown under Inductive and NonInductive Light Conditions JAN KOPCEWICZ With 2 Figures Received January 21, 1972
Summary The high content of estrogens in plants is closely connected with the development of generative organs. Estrogens appear at the period of initiation of inflorescences and reach a maximum at the time of flower development. Two estrogen-like substances have been extracted from Perilla and one from Chenopodium.
Introduction In animals, the oxidation of the C-19 methyl group of androstendione and aromatization of ring A results in the formation of estrogens. Recent experiments indicate that the same reaction sequence also occurs in plants (HEFTMANN, 1968). So estrogens have also been isolated from plant tissues (HEFTMANN, 1963; SINGH et aI., 1969). In previous papers (KOPCEWICZ, 1971; 1972) it has been shown that estrogens, in developing neutral and long-day plants, appear at the time of flower bud formation and reach a maximum at the time of flower development. This paper is a continuation of those studies and examines the quantitative changes in estrogens in typical shortday plants grown under inductive and non-inductive conditions. Materials and Methods The experiments were carried out under controlled conditions in growth chambers (16 or 8 hours light, cool-white fluorescent tubes, light intensity 6500 lx, 23° C in light and 18° C in darkness). Seeds of Perilla ocimoides L. and Chenopodium rubrum L. were germinated in boxes with garden soil in a greenhouse. After eighteen (Perilla) or twenty (Chenopodium) days the seedlings were selected and exposed to long (LD) or short (SD) photoperiod. Material was taken for analysis at the following stages of development. a) Perilla: 1. 10 days after the transfer of plants to growth chamber; 2. 20 days; 3. 25 days; 4. 30 days (the first flower buds of inflorescence were about 2 mm in diam.); 5. 35 days; 6. 40 days (two fully developed flowers); 7. 42 days (four developed flowers); 8. 50 days (the crown of the two oldest flowers fell down); 9. 70 days (the oldest fruits with brown pericarp). The
z. Pflanzenphysiol.
Bd. 67. S. 373-376. 1972.
374
J. KOPCEWICZ
plants produced inflorescences and flowers only in short photoperiod. b) Chenopodium: 1. 20 days after the transfer of plants to long photoperiod; 2. 30 LD; 3. 40 LD; 4. 50 LD; 5. 60 LD; 6. 60 LD + 1 SD; 7. 60 LD + 2 SD; 8. 60 LD + 4 SD (a period of initiation of inflorescences); 9. a) 60 LD + 6 SD, b) 60 LD + 4 SD + 2 LD (first fully developed flowers); 10. a) 60 LD + 14 SD, b) 60 LD + 4 SD + 10 LD (formation of first fruits); 11. a) 60 LD + 24 SD, b) 60 LD + 4 SD + 20 LD. The whole plants deprived of roots were taken for experiments. Estrogens were extracted, fractionated and chromatographed according to the methods previously described (KOPCEWICZ, 1971). For the quantitative determination the Kober colour reaction (NOCKE, 1961) was applied. The readings were corrected for unspecific background colour by applying Ecorr. = 2E515 - (Em + E556 ) (NOCKE, 1961). The content of estrogen-like substances was expressed as flg equivalent of estrone in 100 g of fresh or dry weight. The results were expressed graphically only on a dry weight basis. Data on a fresh weight basis were essentially identical. Results and Discussion The determination of estrogen-like substances in Perilla show that these compounds appear, in plants grown under inductive conditions, in the period of initiation of inflorescences and reach maximum contents at the time of flower development. Estrogens are absent from plants grown in long photoperiod (Fig. 1).
t.fO
--so
:;"
-ii
---LD
'" ~3D
.s
.,c
~ 20
t;
c."
~ 10 ·5 ~
.,.
~ 0
------7
$tage
B
9
of development
Fig. 1. Estrogens content in Perilla ocimoides grown under inductive and non-inductive light conditions. In the case of Chenopodium (Fig. 2) the lack of estrogens in young plants growing on long, non-inductive photoperiod has been stated. Two short photoperiods which induced flowering caused at the same time the increasing of estrogen contents. The transfer of plants back on long photoperiod does not influence the content of estrogens. The plants growing both on long and short photoperiod contain very similar amounts of these compounds (Fig. 2). It seems that estrogens content is closely and substantially connected with the flower development in plants. This hypothesis finds
z. Pjlanzenphysiol. Bd. 67. S. 373-376. 1972.
Estrogens in Short-day Plants
375
~
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--LO
~
0. 30
Q
S!
. .. zo c:.. Ii.. '~/O .
SO
LO
-----50
:/ \
-!: c
I~
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I
.!:;
1t
~
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I
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4
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stage
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9
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af development
Fig. 2. Estrogens content in Chenopodium rubrum grown in long (LD) and short (SD) photoperiod. its confirmation also in previously obtained results (KOPCEWICZ, 1971; 1972). Similarly BENNETT et aI. (1967) stated that the phenolic steroids have been found only in the flowering Haplopappus heterophyllus plants. The present experiments do not answer the question whether the increased estrogen biosynthesis is one of the reasons or the result of plant flowering. There are however data that the inhibitor tris-(2-diethyl-aminoethyl}-phosphate-trihydrochloride (SK & F 7997-A 3), which blocks the synthetic pathway of steroids, supresses floral induction in several plants (BONNER et aI., 1963; EVANS, 1964). The steroidal hormones also have flower promoting (CHOUARD, 1937; CZYGAN, 1962; LESHEM, 1967; KOPCEWICZ, 1970) and sex expression (LOVE and LOVE, 1945; KOPCEWICZ, 1971 a; GAWIENOWSKI et aI., 1971) effects on plants. So it seems possible that the increased steroids biosynthesis is necessary if the flowering process is to take place. The estrogen-like substances extracted from three kilograms of flowering Perilla and from one kilogram of Chenopodium have been compared with standard compounds with respect to their mobilities in different solvents and their fluorescence colours. The results (Table) show the presence of two substances in Perilla and one estrogen-like compound in Chenopodium with fluorescence similar to that of known estrogens_ The localization on the chromatograms and the fluorescence colours suggest that the estrogens extracted from Perilla and Chenopodium probably differ both from standard compounds and from each other. Only Factor 2 (extracted from Perilla) and estrone are similar both in mobility and colour (Table)_ Thus the previous (KOPCEWICZ, 1971; 1972) and present results suggest that plants synthesize various, specific for them estrogen-like substances. This fact seems to be important, explaining at the same time non-reproduction of some physiological effects displayed by steroidal hormones in experiments repeated on different plants. Z. P/lanzenphysiol. Ed. 67. S. 373-376. 1972.
J.
376
KOPCEWICZ
Table 1 RF values, colour and fluorescence of standard estrogens and estrogen-like compounds from Perilla ocimoides and Chenopodium rubrum. Substances Estriol Estradiol-17 fJ Estrone Factor 14) Factor 24) Factor A5) ') (10: 2) 3) 4) 5)
RF (X 100)
I')
2
3
4
8 25 45 18 44 29
8 43 55 30 57 48
21 42 66 35 66 56
71 85 96
n
95 98
Colour2) in visible light
Fluorescence 3)
violet yellow-orange yellow-orange yellow-orange yellow-orange orange
yellow yellow-green yellow-green yellow yellow-green yellow-green
Solvents: 1. light petroleum-CHCI 3 -methanol (40: 10: 3); 2. washed CHCl 3-ethanol 1); 3. 10~/o methanol in benzene; 4. acetone-CH2Cl 2 (3: 7). TLC, 70 (J/o H 2 S0 4. TLC, 70 '0/0 H 2S0 4, u.v.-light. Substances extracted from Perilla ocimoides. Compound extracted from Chenopodium rubrum.
References BENNETT, R. D., E. R. LIEBER, and E. HEFTMANN: Plant Physioi. 42, 973 (1967). BONNER, J., E. HEFTMANN, and J. A. D. ZEEVAART: Plant. Physioi. 38,81 (1963). CHOUARD, P.: Compt. rend. Soc. BioI. 126,509 (1937). CZYGAN, F. C.: Naturwissenschaften 49, 285 (1962). EVANS, L. T.: Aust. J. bioI. Sci. 17,24 (1964). GAWIENOWSKI, A. M., R. W. CHENEY, and H. V. MARSH: Phytochem. 10,2033 (1971). HEFTMANN, E.: Ann. Rev. Plant Physioi. 14,225 (1963). - Lloydia 31, 293 (1968). KOPCEWICZ, J.: Naturwissenschaften 57,136 (1970). - Phytochemistry 10, 1423 (1971). - Z. Pflanzenphysioi. 65,92 (1971 a). - New Phytol. 71, 129 (1972). LESHEM, Y.: Phyton 24, 25 (1967). LOVE, A., and D. LOVE: Ark. Bot. 32, 1 (1945). NOCKE, W.: Biochem. J. 78, 593 (1961). SINGH, H., V. K. KAPOOR, and A. S. CHAWLA: J. Scient. Ind. Res. 28, 339 (1969). Dr. JAN KOPCEWICZ, Department of Plant Physiology, Institute of Biology, Copernicus University, Toruli, Poland.
z.
P/lanzenphysiol. Bd. 67. S. 373-376. 1972.
Estrogens in the Short-day Plants Perilla ocimoides and Chenopodium rubrum Grown under Inductive and NonInductive Light Conditions JAN KOPCEWICZ With 2 Figures Received January 21, 1972
Summary The high content of estrogens in plants is closely connected with the development of generative organs. Estrogens appear at the period of initiation of inflorescences and reach a maximum at the time of flower development. Two estrogen-like substances have been extracted from Perilla and one from Chenopodium.
Introduction In animals, the oxidation of the C-19 methyl group of androstendione and aromatization of ring A results in the formation of estrogens. Recent experiments indicate that the same reaction sequence also occurs in plants (HEFTMANN, 1968). So estrogens have also been isolated from plant tissues (HEFTMANN, 1963; SINGH et aI., 1969). In previous papers (KOPCEWICZ, 1971; 1972) it has been shown that estrogens, in developing neutral and long-day plants, appear at the time of flower bud formation and reach a maximum at the time of flower development. This paper is a continuation of those studies and examines the quantitative changes in estrogens in typical shortday plants grown under inductive and non-inductive conditions. Materials and Methods The experiments were carried out under controlled conditions in growth chambers (16 or 8 hours light, cool-white fluorescent tubes, light intensity 6500 lx, 23° C in light and 18° C in darkness). Seeds of Perilla ocimoides L. and Chenopodium rubrum L. were germinated in boxes with garden soil in a greenhouse. After eighteen (Perilla) or twenty (Chenopodium) days the seedlings were selected and exposed to long (LD) or short (SD) photoperiod. Material was taken for analysis at the following stages of development. a) Perilla: 1. 10 days after the transfer of plants to growth chamber; 2. 20 days; 3. 25 days; 4. 30 days (the first flower buds of inflorescence were about 2 mm in diam.); 5. 35 days; 6. 40 days (two fully developed flowers); 7. 42 days (four developed flowers); 8. 50 days (the crown of the two oldest flowers fell down); 9. 70 days (the oldest fruits with brown pericarp). The
z. Pflanzenphysiol.
Bd. 67. S. 373-376. 1972.
374
J. KOPCEWICZ
plants produced inflorescences and flowers only in short photoperiod. b) Chenopodium: 1. 20 days after the transfer of plants to long photoperiod; 2. 30 LD; 3. 40 LD; 4. 50 LD; 5. 60 LD; 6. 60 LD + 1 SD; 7. 60 LD + 2 SD; 8. 60 LD + 4 SD (a period of initiation of inflorescences); 9. a) 60 LD + 6 SD, b) 60 LD + 4 SD + 2 LD (first fully developed flowers); 10. a) 60 LD + 14 SD, b) 60 LD + 4 SD + 10 LD (formation of first fruits); 11. a) 60 LD + 24 SD, b) 60 LD + 4 SD + 20 LD. The whole plants deprived of roots were taken for experiments. Estrogens were extracted, fractionated and chromatographed according to the methods previously described (KOPCEWICZ, 1971). For the quantitative determination the Kober colour reaction (NOCKE, 1961) was applied. The readings were corrected for unspecific background colour by applying Ecorr. = 2E515 - (Em + E556 ) (NOCKE, 1961). The content of estrogen-like substances was expressed as flg equivalent of estrone in 100 g of fresh or dry weight. The results were expressed graphically only on a dry weight basis. Data on a fresh weight basis were essentially identical. Results and Discussion The determination of estrogen-like substances in Perilla show that these compounds appear, in plants grown under inductive conditions, in the period of initiation of inflorescences and reach maximum contents at the time of flower development. Estrogens are absent from plants grown in long photoperiod (Fig. 1).
t.fO
--so
:;"
-ii
---LD
'" ~3D
.s
.,c
~ 20
t;
c."
~ 10 ·5 ~
.,.
~ 0
------7
$tage
B
9
of development
Fig. 1. Estrogens content in Perilla ocimoides grown under inductive and non-inductive light conditions. In the case of Chenopodium (Fig. 2) the lack of estrogens in young plants growing on long, non-inductive photoperiod has been stated. Two short photoperiods which induced flowering caused at the same time the increasing of estrogen contents. The transfer of plants back on long photoperiod does not influence the content of estrogens. The plants growing both on long and short photoperiod contain very similar amounts of these compounds (Fig. 2). It seems that estrogens content is closely and substantially connected with the flower development in plants. This hypothesis finds
z. Pjlanzenphysiol. Bd. 67. S. 373-376. 1972.
Estrogens in Short-day Plants
375
~
'"
--LO
~
0. 30
Q
S!
. .. zo c:.. Ii.. '~/O .
SO
LO
-----50
:/ \
-!: c
I~
0
I
.!:;
1t
~
•
I
I J
4
8
stage
~
.\ \
9
/0
"
af development
Fig. 2. Estrogens content in Chenopodium rubrum grown in long (LD) and short (SD) photoperiod. its confirmation also in previously obtained results (KOPCEWICZ, 1971; 1972). Similarly BENNETT et aI. (1967) stated that the phenolic steroids have been found only in the flowering Haplopappus heterophyllus plants. The present experiments do not answer the question whether the increased estrogen biosynthesis is one of the reasons or the result of plant flowering. There are however data that the inhibitor tris-(2-diethyl-aminoethyl}-phosphate-trihydrochloride (SK & F 7997-A 3), which blocks the synthetic pathway of steroids, supresses floral induction in several plants (BONNER et aI., 1963; EVANS, 1964). The steroidal hormones also have flower promoting (CHOUARD, 1937; CZYGAN, 1962; LESHEM, 1967; KOPCEWICZ, 1970) and sex expression (LOVE and LOVE, 1945; KOPCEWICZ, 1971 a; GAWIENOWSKI et aI., 1971) effects on plants. So it seems possible that the increased steroids biosynthesis is necessary if the flowering process is to take place. The estrogen-like substances extracted from three kilograms of flowering Perilla and from one kilogram of Chenopodium have been compared with standard compounds with respect to their mobilities in different solvents and their fluorescence colours. The results (Table) show the presence of two substances in Perilla and one estrogen-like compound in Chenopodium with fluorescence similar to that of known estrogens_ The localization on the chromatograms and the fluorescence colours suggest that the estrogens extracted from Perilla and Chenopodium probably differ both from standard compounds and from each other. Only Factor 2 (extracted from Perilla) and estrone are similar both in mobility and colour (Table)_ Thus the previous (KOPCEWICZ, 1971; 1972) and present results suggest that plants synthesize various, specific for them estrogen-like substances. This fact seems to be important, explaining at the same time non-reproduction of some physiological effects displayed by steroidal hormones in experiments repeated on different plants. Z. P/lanzenphysiol. Ed. 67. S. 373-376. 1972.
J.
376
KOPCEWICZ
Table 1 RF values, colour and fluorescence of standard estrogens and estrogen-like compounds from Perilla ocimoides and Chenopodium rubrum. Substances Estriol Estradiol-17 fJ Estrone Factor 14) Factor 24) Factor A5) ') (10: 2) 3) 4) 5)
RF (X 100)
I')
2
3
4
8 25 45 18 44 29
8 43 55 30 57 48
21 42 66 35 66 56
71 85 96
n
95 98
Colour2) in visible light
Fluorescence 3)
violet yellow-orange yellow-orange yellow-orange yellow-orange orange
yellow yellow-green yellow-green yellow yellow-green yellow-green
Solvents: 1. light petroleum-CHCI 3 -methanol (40: 10: 3); 2. washed CHCl 3-ethanol 1); 3. 10~/o methanol in benzene; 4. acetone-CH2Cl 2 (3: 7). TLC, 70 (J/o H 2 S0 4. TLC, 70 '0/0 H 2S0 4, u.v.-light. Substances extracted from Perilla ocimoides. Compound extracted from Chenopodium rubrum.
References BENNETT, R. D., E. R. LIEBER, and E. HEFTMANN: Plant Physioi. 42, 973 (1967). BONNER, J., E. HEFTMANN, and J. A. D. ZEEVAART: Plant. Physioi. 38,81 (1963). CHOUARD, P.: Compt. rend. Soc. BioI. 126,509 (1937). CZYGAN, F. C.: Naturwissenschaften 49, 285 (1962). EVANS, L. T.: Aust. J. bioI. Sci. 17,24 (1964). GAWIENOWSKI, A. M., R. W. CHENEY, and H. V. MARSH: Phytochem. 10,2033 (1971). HEFTMANN, E.: Ann. Rev. Plant Physioi. 14,225 (1963). - Lloydia 31, 293 (1968). KOPCEWICZ, J.: Naturwissenschaften 57,136 (1970). - Phytochemistry 10, 1423 (1971). - Z. Pflanzenphysioi. 65,92 (1971 a). - New Phytol. 71, 129 (1972). LESHEM, Y.: Phyton 24, 25 (1967). LOVE, A., and D. LOVE: Ark. Bot. 32, 1 (1945). NOCKE, W.: Biochem. J. 78, 593 (1961). SINGH, H., V. K. KAPOOR, and A. S. CHAWLA: J. Scient. Ind. Res. 28, 339 (1969). Dr. JAN KOPCEWICZ, Department of Plant Physiology, Institute of Biology, Copernicus University, Toruli, Poland.
z.
P/lanzenphysiol. Bd. 67. S. 373-376. 1972.