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Physiological Effects of Steviol
Departemento de Fisiologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, 13 100 Campinas, SP, Brasil
Physiological Effects of Steviol I. F. M.
VALlO
and
ROSELY
F.
ROCHA
With 2 Figures Received November 7, 1975 . Accepted November 11, 1975
Summary Steviol, a plant terpenoid, shows gibberellin-like actiVity in the lettuce and cucumber hypocotyl elongation tests and on growth of bean plants. In some other species steviol is ineffective or even shows an effect opposite to that of gibberellin.
Key words: Steviol, gibberellin, gibberellin-like substance, elongation tests.
Introduction Steviol, a product of enzymatic hydrolysis of the stevioside from Stevia rebaudiana Bertoni is a tetra cyclic diterpene structurally related to the gibberellins. This structural similarity suggested a gibberellin-like activity of steviol (RUDDAT et aI., 1963). Other evidence for this hypothesis is the similar biosynthetic pathway of gibbereIIins and steviol (HANSON and WHITE, 1968). Thus, steviol has a chemical structure and biosynthetic pathway similar to that of gibberellins and in some species shows gibberellin-like activity whereas in others it is ineffective. The aim of this work was to demonstrate the gibberellin-like activity of steviol in some species other than S. rebaudiana.
Material and Methods Stevia rebaudiana Bert. from Paraguay was cultivated in the Instituto de Boranica of S. Paulo. Cucumis sativus L. cv. Meio Longo, Lactuca sativa cv. Gigante 1-1797 and Phaseolus vulgaris cv. Pintado were used in the bioassays. Bioassays: the lettuce hypocotyl assay was carried out according to FRANKLAND and WAREING (1960). A similar procedure was used when this bioassay was performed in darkness. The cucumber hypocotyl assay was based on BRIAN et al. (1964). Bean seedlings were treated with an aqueous solution of steviol as a droplet on the 6th and 7th day after planting (total of 50 flg per plant). 20 ml of a 2000 flg i ml solution of (2-chloroethyl) trimethyl ammonium chloride (Ccq were applied to each pot as a soil drench on the day and the next after sowing the seeds. The bean seedlings were kept in the glasshouse for 15 days. Dry weight was determined after a period of 24 h at 100 ° C.
Z. PJlanzenphysiol. Bd.
78.
S. 90-94. 1976.
Physiological Effects of Steviol
91
Extraction: extraction of stevioside was based on the method of BRID EL and LAVIEILLE (1931 a, b). Steviol was then obtained by enzymatic hydrolysis of the stevioside (RUDDAT et al., 1965).
Results and Discussion
Fig. 1 shows the stimulating effect of steviol on cucumber hypocotyl elongation. In this experiment concentrations higher than 10 ug/ml are statistically significant at 5 % level to the control. 120
c 0
a
0 L
CJ'~
C
C
."
0 u
0
110
>-- -
-0
lS d
0
j5 0/0
a ou ____
0-0
>--
L
100 0
I
2
steviol log }lg /ml
Fig. 1: Effect of steviol on the elongation of cucumber hypocotyl.
The lettuce hypocotyl bioassay was carried out under continuous light or in darkness. Steviol and gibberellic acid (GA 3) were applied to the lettuce seedlings at 1, 10 and 100 ug/ ml. In darkness GA 3 did not affect hypocotyl growth while steviol enhanced growth. Under continuous light both GA 3 and steviol stimulated elongation of the hypocotyls, GA 3 being more effective than steviol (Fig. 2). Steviol showed gibberellin-like activity on the growth of cucumber and lettuce hypocotyl. These results do not agree with those of NITSCH and NITSCH (1965) for lettuce and of KATSUMI et al. (1964) for cucumber. These discrepancies may be due to different cultivars used. A promotive effect of steviol on specific bioassays for gibberellins has been demonstrated before for dwarf corn d-5 and an-l (RUDDAT et aI., 1963; KATSUMI et aI., 1964). Table 1 shows the effect of steviol and CCC on bean plants. Steviol promoted elongation of the primary leaf petioles. Elongation of the hypocotyl, epicotyl and first internode were statistically not significant. CCC strongly inhibited the growth of the plants. This inhibitory effect was partially reverted by steviol. In relation to dry weight steviol promoted growth of the epicotyl, primary leaf petioles, stem (above epicotyl), blade of primary leaf, first leaf and roots. CCC inhibited most of the growth and steviol reverted the CCC inhibitory effect of stem (above epicotyl) and first leaf (Table 2).
z. Pjlanzenphysiol. Bd. 78. S. 90-94.
1976.
92
I. F. M.
VALlO
and
ROSELY
F.
ROCHA
250 o
L.
C o
u
'0 200
l
is d SOlo
o o
---c
Q
g, c
150
~
'"
>-.
+-'
o
u
~IOO
>-.
L
o
2 log jJg/ml
Fig. 2: Effect of GA3 and steviol on the elongation of lettuce hypocotyl GA3 darkness; (.) steviollight; (D) steviol darkness.
Table 1: Effect of steviol, CCC and steviol Growth (em) hypocotyl epicotyl petiole (primary leaf) 1st internode
Ce)
GA3 light; (0)
+ CCC on elongation of bean seedlings. + CCC
Control
Steviol
CCC
9.0 6.3
9.5 6.4
4.6""· 1.7',."
4.8 2.2aa
6.1 7.0
6.4"· 7.7
2.4"·"· 0.2"'"
O.5 aa
Steviol
2.9 aa
". - statistically significant at 5 % level to the control. •. ". - statistically significant at 1 % level to the control. aa - statistically significant at 1 % level to the CCC treatment.
Thus, steviol promoted growth of bean plants by increasing elongation and dry weight. Some of these effects are similar to those caused by gibberellic acid (FELIPPE, 1969). Similar gibberellin-like activity is shown by the reversion of the inhibitory effect caused by ccc. RUDDAT (1968) observed promotive effect of steviol on growth of dwarf peas treated with CCC grown in darkness. We obtained similar results although not as conspicuous as reported by RUDDAT (1968). Nevertheless in other experiments steviol was ineffective or even showed an inhibitory effect. In S. rebaudiana GA 3 induces 100 0/0 of parthenocarpic fruits but steviol was almost ineffective. GA 3 also stimulates stem elongation in S. rebau-
z. Pjlanzenphysiol. Ed. 78. S. 90-94. 1976.
Physiological Effects of Steviol Table 2: Effect of steviol, CCC and steviol Growth (mg) hypocotyl epicotyl petiole (primary leaf) stem (above epicotyl) primary leaf (blade) first leaf root
93
+ CCC on dryweight of bean seedlings.
Control
Steviol
52.3 26.6
59.1 31.P
10.9
CCC
Steviol
+ CCC
8.5"0"-0
27.1 9.9
13.2"0"0
6.6"0"0
7.0
22.2
31.5*,,0
6.0"0"0
9.9 aa
71.3 42.5 62.2
89.1"-0"0 56.6"0"0 78.4"0"0
25. 3~· "0
75 .6 14.4*" 64.3
76.7 22.7aa 70.0
" - statistically significant at 5 % level to the control. "0"0 _ statistically significant at 1 % level to the control. an - statistically significant at 1 Ofo level to the CCC treatment.
diana while steviol shows an opposite effect. GA 3 induces bolting in Kalanchoe gastonis-bonnieri under short or long days. Besides bolting GA 3 induces flowering in short days. Steviol had no effect at all on this species. It seems that steviol can act as a gibberellin or not. When it possesses a gibberellinlike activity this effect could be due to its own effect or due to gibberellins biosynthesized from it. That gibberellins are biosynthesized from steviol was suggested by KATSUMI et al. (1964). The lack of activity of steviol in certain species might be attributed to the biosynthesis of certain gibberellins inactive in these species or to the lack of enzymes responsible for the conversion of steviol to gibberellins. The fact that the pathways for biosynthesis of gibberellins and steviol are very similar and the gibberellin activity known in several species suggest that steviol can act as a gibberellin or can be a precursor of gibberellins. Acknowledgements The authors wish to thank Drs. G. M. FELIPPE and L. SODEK for reading the manuscript and helpful discussion. Thanks are also due to the Fundas:ao de Amparo a Pes qui sa do Estado de S. Paulo (FAPESP) for financial support. References BRIAN, P. W., H . G. HEMMING and D. LOWE: Ann. Bot. 28, 369 (1964). BRIDEL, L. M., and R. LAVIEILLE: Bdl. Soc. Chim. bioI. 13,636 (1931 a). - - J. Pharm. Chim. 14, 99 (1931 b) . FELIPPE, G. M.: Phyton 26, 3 (1969). FRANKLAND, B., and P. F. WAREING: Nature 185,255 (1960) .
z. Pflanzenphysiol. Bd. 78. S. 90-94.
1976.
94
1. F. M. VALlO and ROSELY F. ROCHA
HANSON, J. R., and A. F. WHITE: Phytochemistry 7, 595 (1968). KATSUMI, M., B. O. PHINNEY, P. R. JEFFERIS and C. A. HENRICK: Science 144, 849 (1964). NITSCH, J. P., and C. NITSCH: Ann. Physio!. Veg. 7, 259 (1965). RUDDAT, M.: In «Biochemistry and Physiology of Plant Growth Substances», ed. F. WIGHTMAN and R. G. SETTERFIELD (1968). RUDDAT, M., A. LANG and E. MOSETTIG: Naturwissenschaften 50, 23 (1963) RUDDAT, M., E. HEFTMANN and A. LANG: Arch. Biochem. Biophys. 110, 496 (1965).
1. F. M. VALlO and ROSELY F. ROCHA, Departemento de Fisiologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, 13100 Campinas, SP, Brasil.
Z. Pjlanzenphysiol. Ed. 78. S. 90-94. 1976.
Physiological Effects of Steviol I. F. M.
VALlO
and
ROSELY
F.
ROCHA
With 2 Figures Received November 7, 1975 . Accepted November 11, 1975
Summary Steviol, a plant terpenoid, shows gibberellin-like actiVity in the lettuce and cucumber hypocotyl elongation tests and on growth of bean plants. In some other species steviol is ineffective or even shows an effect opposite to that of gibberellin.
Key words: Steviol, gibberellin, gibberellin-like substance, elongation tests.
Introduction Steviol, a product of enzymatic hydrolysis of the stevioside from Stevia rebaudiana Bertoni is a tetra cyclic diterpene structurally related to the gibberellins. This structural similarity suggested a gibberellin-like activity of steviol (RUDDAT et aI., 1963). Other evidence for this hypothesis is the similar biosynthetic pathway of gibbereIIins and steviol (HANSON and WHITE, 1968). Thus, steviol has a chemical structure and biosynthetic pathway similar to that of gibberellins and in some species shows gibberellin-like activity whereas in others it is ineffective. The aim of this work was to demonstrate the gibberellin-like activity of steviol in some species other than S. rebaudiana.
Material and Methods Stevia rebaudiana Bert. from Paraguay was cultivated in the Instituto de Boranica of S. Paulo. Cucumis sativus L. cv. Meio Longo, Lactuca sativa cv. Gigante 1-1797 and Phaseolus vulgaris cv. Pintado were used in the bioassays. Bioassays: the lettuce hypocotyl assay was carried out according to FRANKLAND and WAREING (1960). A similar procedure was used when this bioassay was performed in darkness. The cucumber hypocotyl assay was based on BRIAN et al. (1964). Bean seedlings were treated with an aqueous solution of steviol as a droplet on the 6th and 7th day after planting (total of 50 flg per plant). 20 ml of a 2000 flg i ml solution of (2-chloroethyl) trimethyl ammonium chloride (Ccq were applied to each pot as a soil drench on the day and the next after sowing the seeds. The bean seedlings were kept in the glasshouse for 15 days. Dry weight was determined after a period of 24 h at 100 ° C.
Z. PJlanzenphysiol. Bd.
78.
S. 90-94. 1976.
Physiological Effects of Steviol
91
Extraction: extraction of stevioside was based on the method of BRID EL and LAVIEILLE (1931 a, b). Steviol was then obtained by enzymatic hydrolysis of the stevioside (RUDDAT et al., 1965).
Results and Discussion
Fig. 1 shows the stimulating effect of steviol on cucumber hypocotyl elongation. In this experiment concentrations higher than 10 ug/ml are statistically significant at 5 % level to the control. 120
c 0
a
0 L
CJ'~
C
C
."
0 u
0
110
>-- -
-0
lS d
0
j5 0/0
a ou ____
0-0
>--
L
100 0
I
2
steviol log }lg /ml
Fig. 1: Effect of steviol on the elongation of cucumber hypocotyl.
The lettuce hypocotyl bioassay was carried out under continuous light or in darkness. Steviol and gibberellic acid (GA 3) were applied to the lettuce seedlings at 1, 10 and 100 ug/ ml. In darkness GA 3 did not affect hypocotyl growth while steviol enhanced growth. Under continuous light both GA 3 and steviol stimulated elongation of the hypocotyls, GA 3 being more effective than steviol (Fig. 2). Steviol showed gibberellin-like activity on the growth of cucumber and lettuce hypocotyl. These results do not agree with those of NITSCH and NITSCH (1965) for lettuce and of KATSUMI et al. (1964) for cucumber. These discrepancies may be due to different cultivars used. A promotive effect of steviol on specific bioassays for gibberellins has been demonstrated before for dwarf corn d-5 and an-l (RUDDAT et aI., 1963; KATSUMI et aI., 1964). Table 1 shows the effect of steviol and CCC on bean plants. Steviol promoted elongation of the primary leaf petioles. Elongation of the hypocotyl, epicotyl and first internode were statistically not significant. CCC strongly inhibited the growth of the plants. This inhibitory effect was partially reverted by steviol. In relation to dry weight steviol promoted growth of the epicotyl, primary leaf petioles, stem (above epicotyl), blade of primary leaf, first leaf and roots. CCC inhibited most of the growth and steviol reverted the CCC inhibitory effect of stem (above epicotyl) and first leaf (Table 2).
z. Pjlanzenphysiol. Bd. 78. S. 90-94.
1976.
92
I. F. M.
VALlO
and
ROSELY
F.
ROCHA
250 o
L.
C o
u
'0 200
l
is d SOlo
o o
---c
Q
g, c
150
~
'"
>-.
+-'
o
u
~IOO
>-.
L
o
2 log jJg/ml
Fig. 2: Effect of GA3 and steviol on the elongation of lettuce hypocotyl GA3 darkness; (.) steviollight; (D) steviol darkness.
Table 1: Effect of steviol, CCC and steviol Growth (em) hypocotyl epicotyl petiole (primary leaf) 1st internode
Ce)
GA3 light; (0)
+ CCC on elongation of bean seedlings. + CCC
Control
Steviol
CCC
9.0 6.3
9.5 6.4
4.6""· 1.7',."
4.8 2.2aa
6.1 7.0
6.4"· 7.7
2.4"·"· 0.2"'"
O.5 aa
Steviol
2.9 aa
". - statistically significant at 5 % level to the control. •. ". - statistically significant at 1 % level to the control. aa - statistically significant at 1 % level to the CCC treatment.
Thus, steviol promoted growth of bean plants by increasing elongation and dry weight. Some of these effects are similar to those caused by gibberellic acid (FELIPPE, 1969). Similar gibberellin-like activity is shown by the reversion of the inhibitory effect caused by ccc. RUDDAT (1968) observed promotive effect of steviol on growth of dwarf peas treated with CCC grown in darkness. We obtained similar results although not as conspicuous as reported by RUDDAT (1968). Nevertheless in other experiments steviol was ineffective or even showed an inhibitory effect. In S. rebaudiana GA 3 induces 100 0/0 of parthenocarpic fruits but steviol was almost ineffective. GA 3 also stimulates stem elongation in S. rebau-
z. Pjlanzenphysiol. Ed. 78. S. 90-94. 1976.
Physiological Effects of Steviol Table 2: Effect of steviol, CCC and steviol Growth (mg) hypocotyl epicotyl petiole (primary leaf) stem (above epicotyl) primary leaf (blade) first leaf root
93
+ CCC on dryweight of bean seedlings.
Control
Steviol
52.3 26.6
59.1 31.P
10.9
CCC
Steviol
+ CCC
8.5"0"-0
27.1 9.9
13.2"0"0
6.6"0"0
7.0
22.2
31.5*,,0
6.0"0"0
9.9 aa
71.3 42.5 62.2
89.1"-0"0 56.6"0"0 78.4"0"0
25. 3~· "0
75 .6 14.4*" 64.3
76.7 22.7aa 70.0
" - statistically significant at 5 % level to the control. "0"0 _ statistically significant at 1 % level to the control. an - statistically significant at 1 Ofo level to the CCC treatment.
diana while steviol shows an opposite effect. GA 3 induces bolting in Kalanchoe gastonis-bonnieri under short or long days. Besides bolting GA 3 induces flowering in short days. Steviol had no effect at all on this species. It seems that steviol can act as a gibberellin or not. When it possesses a gibberellinlike activity this effect could be due to its own effect or due to gibberellins biosynthesized from it. That gibberellins are biosynthesized from steviol was suggested by KATSUMI et al. (1964). The lack of activity of steviol in certain species might be attributed to the biosynthesis of certain gibberellins inactive in these species or to the lack of enzymes responsible for the conversion of steviol to gibberellins. The fact that the pathways for biosynthesis of gibberellins and steviol are very similar and the gibberellin activity known in several species suggest that steviol can act as a gibberellin or can be a precursor of gibberellins. Acknowledgements The authors wish to thank Drs. G. M. FELIPPE and L. SODEK for reading the manuscript and helpful discussion. Thanks are also due to the Fundas:ao de Amparo a Pes qui sa do Estado de S. Paulo (FAPESP) for financial support. References BRIAN, P. W., H . G. HEMMING and D. LOWE: Ann. Bot. 28, 369 (1964). BRIDEL, L. M., and R. LAVIEILLE: Bdl. Soc. Chim. bioI. 13,636 (1931 a). - - J. Pharm. Chim. 14, 99 (1931 b) . FELIPPE, G. M.: Phyton 26, 3 (1969). FRANKLAND, B., and P. F. WAREING: Nature 185,255 (1960) .
z. Pflanzenphysiol. Bd. 78. S. 90-94.
1976.
94
1. F. M. VALlO and ROSELY F. ROCHA
HANSON, J. R., and A. F. WHITE: Phytochemistry 7, 595 (1968). KATSUMI, M., B. O. PHINNEY, P. R. JEFFERIS and C. A. HENRICK: Science 144, 849 (1964). NITSCH, J. P., and C. NITSCH: Ann. Physio!. Veg. 7, 259 (1965). RUDDAT, M.: In «Biochemistry and Physiology of Plant Growth Substances», ed. F. WIGHTMAN and R. G. SETTERFIELD (1968). RUDDAT, M., A. LANG and E. MOSETTIG: Naturwissenschaften 50, 23 (1963) RUDDAT, M., E. HEFTMANN and A. LANG: Arch. Biochem. Biophys. 110, 496 (1965).
1. F. M. VALlO and ROSELY F. ROCHA, Departemento de Fisiologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas, 13100 Campinas, SP, Brasil.
Z. Pjlanzenphysiol. Ed. 78. S. 90-94. 1976.