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The Effect of Arachidonic Acid, Prostaglandins and Inhibitors of Prostaglandin Synthetase, on the Flowering of Excised Pharbitis nil Shoot Apices under Different Photoperiods
Margaretha Mes Institute for Plant Physiology, University of Pretoria, Pretoria, Republic of South Africa
The Effect of Arachidonic Acid, Prostaglandins and Inhibitors of ProstagIandin Synthetase, on the Flowering of Excised Pharbitis nil Shoot Apices under Different Photoperiods E. G. GROENEWALD and J. H. VISSER With 6 Figures Received 12 February 1978 . Accepted 6 March 1978
Summary Arachidonic acid and PGE 1 (10-4 M) hastened the time to flowering of Pharbitis nil plantlets which developed from excised shoot apices under inductive conditions (short days). Gentisic acid, acetylsalicylic acid, salicylic acid and oleic acid, all known inhibitors of prostaglandin synthetase, inhibited flowering of plantlets under inductive conditions. Arachidonic acid and PGE 1, PGE 2, PGF 1a, PGF 2a, PGA1, PGA 2, PGB 1, and PGB 2, (10-4 M, 10-5 M and 10-6 M) did not induce flowering in plantlets which were kept under long days. However, leaf blade growth was reduced by the treatments. A hypothetical scheme is presented for the possible regulation of flowering by prostaglandins and phenolic acids (salicylic acid). Key wards: Pharbitis nil; flawering; prastaglandins; inhibitors.
Introduction
In a previous paper (GROENEWALD and VISSER, 1974) it was reported that certain inhibitors of prostagiandin (PG) biosynthesis inhibited flowering of intact Pharbitis nil plants. Pharbitis nil is a short day plant. At the time when the above mentioned experiments were conducted it was not certain whether prostaglandins occurred naturally in plants. However, recently PGA 1 was discovered in the onion (Allium cepa) by ATTREP et al (1973) and MIYARES CAO and MENENDEZ CEPERO (1976) reported the occurrence of prostaglandin-like compounds in Saccharum officinarum, Musa paradisiaca and Cocos nucifera. Also, in prelimenary experiments with aseptic excised Pharbitis shoot apices, GROENEWALD and VISSER (unpublished results) found that applied labelIed arachidonic acid was converted to probably PGE2, PGF2 a, PGA2 and PGB 2 • In this paper we report on the effect of applied archidonic acid, PGEt, gentlslC acid, acetylsalicylic acid, salicylic acid and oleic acid, on flowering of aseptic excised Z. Pflanzenphysial. Bd. 88. S. 423-429. 1978.
424
E. G. GROENEWALD and ]. H. VISSER
Pharbitis shoot apices kept under short day conditions (8 hr light - 16 hr darkness). The concentration of the compounds was 10- 4 M. Also, on the effect of PGE h PGE 2 , PGF 1 a, PGF 2a, PGA h PGA 2, PGB 1 and PGB 2 and arachidonic acid on flowering of aseptic excised Pharbitis shoot apices kept under long days (8 hr darkness - 16 hr light). The concentrations of the compounds were 10- 4 M, 10- 5 M and 10-6 M. Material and Methods Pharbitis nil Chois. Strain «Violet » seeds were treated with conc. H 2S0 4 for 45 min., rinsed with dist. H 20 and sterilized in 70 % EtOH for 10 min., followed by 60 min. in 10 Ofo Ca(OClh and finally in a mixture of 0.1 Ofo HgCl 2 and 0.1 Ofo sodium lauryl sulphate for 10 min. The sterilized seeds were immediately rinsed in 4 changes of sterile dist. H 20. The sterilized seeds were placed onto wet cotton wool contained in previously sterilized 250 ml erlenmeyer flasks. The flasks containing the seeds were kept for 3 days in a constant tempo room (27 °C) and under continuous light (3450 lux) supplied by cool white fluorescent tubes. At the end of this period the Pharbitis seedlings were about 4 cm high, and using aseptic techniques the hypocotyls were cut 2 cm below the growing points and the cotyledons were removed. The plant segments obtained in this way were sterilized for 5 min. in 5 % Ca(OClh and rinsed with sterile dist. H 20. This second sterilization treatment was necessary because of a fungus that was hard to get rid of and was probably present inside the testa of the seed. The sterilized plant segments were trimmed at the cut edges and placed upright in 5 ml agar nutrient medium contained in test tubes (23 mm X 195 mm). The composition of the nu trient medium was mineral salts (LINSMAIER and SKOOG, 1965), sucrose 3
Fig. 1: A Pharbitis shoot segment (apex) in nutrient agar medium in a test tube.
z.
Pjlanzenphysiol. Bd. 88. S. 423-429. 1978.
The effect of arachidonic acid
425
ml of a sodium carbonate solution (0.2 mg/mI H 20). The pH was measured with pHydrion paper and adjusted to between 6 and 7.5. The dissolved compound was then filtered through a Millipore filter (0.45 ftm pore size). The filter sterilized dissolved compound (0.2 ml!5 ml agar medium) was added to the autoclaved agar nutrient medium when it reached a tempo of 40°-45 oe and it was weIl mixed and aIlowed to set. The shoot apices were usuaIly kept in nutrient ag ar medium without any of the test compounds added, for 3 days, to make certain that the apices were not infected with fungus. The apices were kept at 25 oe and under long day conditions (8 hr darkness - 16 hr light). At the end of this period a smaIl segment was cut off (ca. 5 mm) from the cut end of the hypocotyl of each plant segment (apex) and the apices were transferred to agar nutrient medium containing the test compound (Fig. 1). The test tubes containing the shoot apices were kept at 25 oe and either under long day conditions (8 hr darkness - 16 hr light) or short day conditions (8 hr light - 16 hr darkness). The light intensity was 8600 lux and was supplied by fluorescent tubes and incandescent lamps.
Results and Discussion
In the experiment where arachidonic acid, PGEt, gentisic acid, ac'etylsalicylic acid, salicylic acid and oleic acid were applied to excised shoot apices under inductive conditions (short days) it was found that arachidonic acid and PGE t hastened flower formation by 29 days and 28 days respectively as compared with controls. A Pharbitis plantlet with a terminal flower, in a test tube, is depic'ted in Fig. 2. The benzoic acid derivatives (gentisic acid, acetylsalicylic acid and salicylic acid) a11 inhibited flowering completely. The results are summarized in Table 1.
Fig. 2: A Pharbitis plantlet which developed from an apex in a test tube. Note the flower. Z. Pflanzenphysiol. Bd. 88. S. 423-429. 1978.
426
E. G. GROENEWALD and
J.
H. VISSER
Table 1: EHect of various chemical compounds (10-4 M) on flowering of excised shoot apices of Pharbitis kept under inductive (8 hr light - 16 hr darkness) conditions. Five replicates were used per treatment. Treatment
Number of plants that flowered
Flowering first noticed (days after start of experiments)
Control Arachidonic acid PGE 1 Gentisic acid Acetylsalicylic acid Salicylic acid Oleic acid
5 5 3
45 16 17
o o
o
o
In the experiment where arachidonic acid, PGEt, PGE 2, PGF1a, PGF 2a, PGAt, PGA 2 , PGB 1 and PGB 2 were applied to excised apices under non-inductive (long day) conditions it was found that none of the treatments induced flower formation. However, in most of the treatments the higher concentrations (10- 4 M and 10-5 M) caused a reduction in leaf bl ade growth of the plantlets. Three types of responses were encountered, namely, A, Band C and they are depicted in Fig. 3, 4 and 5 respectively. The results are summarized in Table 2.
Fig.3
Fig.4
Fig.5
Fig. 3: EHect of prostagiandin (10-4 M) on the growth of a shoot apex of Pharbitis under long day conditions. Leaf growth was drastically reduced (see text for details). Fig. 4: EHect of prostagiandin (10-5 M) on the growth of a shoot apex of Pharbitis under long day conditions. The shape of the leaf blade was altered by the treatment (arrowhead shape) (see text for details). Fig. 5: EHect of prostagiandin (10- 6 M) on the growth of a shoot apex of Pharbitis under long day conditions. Plantlet similar to untreated (control) apex (see text for details). Z. Pjlanzenphysiol. Bd. 88. S. 423-429. 1978.
The effect of arachidonic acid
427
Table 2: Summary of results obtained with the addition of arachidonic acid and different prostaglandins to the nutrient agar in which shoot apices of Pharbitis were grown under long days (8 hr darkness - 16 hr light). Responses to the added substances are designated A, Band C and are depicted by Figs. 3, 4 and 5. The fractions indicate the number of apices out of the total of replicates that gave a certain response. Controls gave response C.
Treatment
Reaction to different concentrations 10-sM 10-4M 10-6 M
Arachidonic acid PGE1 PGE 2 PGF 1a PGF 2a PGA 1 PGA2 PGB 1 PGB 2
A 4/S A 2/r. A 2/S B 4/S A 3/S A 2/ S A 1/2 B 4/4 B 3/.
B B B B B B B B B
3/S 4/. 3/S 3/r. 4/S 31" 3/4 3/3 4/4
C C C B C C C C C
3/3
s/s s/s 1,s /s
4/r. 4/. 5/S 4/4
It is clear from the results obtained in the short day experiment that both arachidonic acid and PGE1 hastened flowering. Arachidonic acid is aprecursor for PGE2 and PGF2 a. The benzoic acid derivatives, namely, gentisic acid, acetylsalicylic acid and salicylic acid are all inhibitors of prostagiandin synthesis. Of the three acids gentisic acid is the most potent inhibitor of prostagiandin synthetase followed by acetylsalicylic acid and salicylic acid (FLOWER, 1974). Another benzoic acid derivative namely, gallic acid was reported (PRYCE, 1972) to be a natural inhibitor of flowering in the short day plant Kalanchoe blossfeldiana. It was, however, found by CLELAND and A]AMI (1974) that honeydew produced by an aphid when feeding on flowering or vegetative plants of the short day plant Xanthium strumarium produced an active substance capable of inducing flowering in the long day plant Lemna gibba G3. The ac'tive substance was found to be salicylic acid. This is in contrast to our findings with Pharbitis nil. Oleic acid, a fatty acid, is also an inhibitor of prostagiandin synthesis (PACE-AsCIAK and WOLFE, 1968). The nature of inhibition is probably competitive. In the experiments conducted under long day conditions the Pharbitis plantlets could not be induced to flower. Since the plantlets produced leaves it is possible that production of natural inhibitors of flowering could have been produced thus preventing flowering. In this regard DE FOSSARD (1974) advocates that specimens should be grown under inductive conditions in order to avoid natural inhibitor formation. Specimens should also be defoliated in order to reduce the quantity of natural florigen produc'tion under the conditions of the test and further specimens should be reduced to the tissue which responds to natural florigens, i.e. the shoot apical meristem in order to avoid indirect responses of applied substances. On the evidence obtained thus far one can put forward a hypothetical scheme for the control of flowering by prostaglandins and phenolic acids as depicted in Fig. 6.
z.
Pjlanzenphysiol. Bd. 88. S. 423-429. 1978.
428
E. G. GROENEWALD and
J.
H. VISSER
Pathways 01 biosynthesis ot prostaglandins and a few ph.nolic acids which may play a rol. in flowaring of Pharbitis.
GIUrSe
Phospholipids
1
Phospholipase H
~
C-20 unsaturated Fatty acids H H H Arachidonic acid Shikimic aci 8,11.14-Eicosatrienoic acid 5.8.11.14,17'Eicosapentaenoic acid
Qc~~-COOH
PG synthetase iCiH2 I-Phenylalanine
r PAL
Prostaglandins
1P
Flowering
Inhibitors ar PGsynthetase and Flowerlng o-Äcetylsallcyllc acid Sallcyllc acid Gentlslc acid Pt1enylbutazone Indomethacln Nfflumlcacld
ry-~-COOH t-Cinnamlc acid
\
,/
(}
'=&
7
~OOH Salicylic acid
Fig. 6: A hypothetical scheme depicting the possible interrelationship between prostaglandins and phenolic inhibitors (salicylic acid) on flowering of Pharbitis nil.
There is chromatographie evidence that salicylic acid (GROENEWALD and VISSER, unpublished results) amongst other phenolic acids, is produced in the cotyledons of Pharbitis nil and it is possible that salicylic acid could be a natural inhibitor of flowering in Pharbitis. In this regard it is interesting to note that phenolic acids arise from the action of phenyl alanine ammonia-lyase (PAL) and that light has a stimulatory effect on the enzyme, and thus the production of phenolic acids. It is thus possible that under long day conditions relatively large amounts of phenolic acids may be produced, as was found by PRYCE (1972) in the case of gallic acid, and this may be the cause of flower inhibition, sinc'e a number of phenolic acids are kno'wn inhibitors of prostagiandin synthetase (FLOWER, 1974). Acknowledgements We thank Dr. J. E. PIKE, Upjohn, Kalamazoo, Mich., U.S.A. for the prostaglandins used in this study. We also thank the C.S.I.R. for financial assistance and the Margaretha Mes Institute, University, of Pretoria, for the facilities provided. Z. Pjlanzenphysiol. Bd. 88. S. 423-429. 1978.
The effect of arachidonic acid
429
References ATTREP, K. A., J. M. MARIANI, Jr., and M. ATTREP, Jr.: Search for prostagiandin Al in onion. Lipids 8, 484-486 (1973). CLELAND, C. F. and A. AJAMI: Identification of the flower-inducing factor isolated from aphid honeydew as being salicylic acid. Plant Physiol. 54, 904-906 (1974). DE FOSSARD, R. A.: Flower initiation in tissue and organ cultures. In: H. E. STREET (Ed.). Tissue culture and Plant Science, pp. 193-212 Academic Press, London and New York, 1974. FLOWER, R. J.: Drugs which inhibit prostagiandin biosynthesis. Pharmacol. Rev. 26, (1), 33-67 (1974). GROENEWALD, E. G. and J. H. VISSER: The effect of certain inhibitors of prostagiandin biosynthesis on flowering of Pharbitis nil. Z. Pflanzenphysiol. 71, 67-70 (1974). LINsMAIER, ELFRIEDE, M and F. SKOOG: Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant 18, 100-127 (1965). MIYARES CAO, C. M. and E. MENENDEZ CEPERO: Identification of prostaglandinlike substances in plants. In: B. SAMUELSSON and R. PAOLETTI (Eds.) Advances in prostagiandin and thromboxane research p. 877. Vol. 2., Raven Press, New York, 1976. PACE-AsCIAK, C. and L. S. WOLFE: Inhibition of prostagiandin synthesis by oleic, linoleic and linolenic acids. Biochim. Biophys. Acta 152, 784-787 (1968). PRYCE, R. J.: Gallic acid as a natural inhibitor of flowering in Kalanchoe blossfeldiana. Phytochemistry 11, 1911-1918 (1972). E. G. GROENEWALD, Department of Botany, University of the O. F. S. Bloemfontein 9300, South Africa. J. H. VISSER, Department of Botany, University of Pretoria, Pretoria, 0002, South Africa.
z.
Pflanzenphysiol. Bd. 88. S. 423-429. 1978.
The Effect of Arachidonic Acid, Prostaglandins and Inhibitors of ProstagIandin Synthetase, on the Flowering of Excised Pharbitis nil Shoot Apices under Different Photoperiods E. G. GROENEWALD and J. H. VISSER With 6 Figures Received 12 February 1978 . Accepted 6 March 1978
Summary Arachidonic acid and PGE 1 (10-4 M) hastened the time to flowering of Pharbitis nil plantlets which developed from excised shoot apices under inductive conditions (short days). Gentisic acid, acetylsalicylic acid, salicylic acid and oleic acid, all known inhibitors of prostaglandin synthetase, inhibited flowering of plantlets under inductive conditions. Arachidonic acid and PGE 1, PGE 2, PGF 1a, PGF 2a, PGA1, PGA 2, PGB 1, and PGB 2, (10-4 M, 10-5 M and 10-6 M) did not induce flowering in plantlets which were kept under long days. However, leaf blade growth was reduced by the treatments. A hypothetical scheme is presented for the possible regulation of flowering by prostaglandins and phenolic acids (salicylic acid). Key wards: Pharbitis nil; flawering; prastaglandins; inhibitors.
Introduction
In a previous paper (GROENEWALD and VISSER, 1974) it was reported that certain inhibitors of prostagiandin (PG) biosynthesis inhibited flowering of intact Pharbitis nil plants. Pharbitis nil is a short day plant. At the time when the above mentioned experiments were conducted it was not certain whether prostaglandins occurred naturally in plants. However, recently PGA 1 was discovered in the onion (Allium cepa) by ATTREP et al (1973) and MIYARES CAO and MENENDEZ CEPERO (1976) reported the occurrence of prostaglandin-like compounds in Saccharum officinarum, Musa paradisiaca and Cocos nucifera. Also, in prelimenary experiments with aseptic excised Pharbitis shoot apices, GROENEWALD and VISSER (unpublished results) found that applied labelIed arachidonic acid was converted to probably PGE2, PGF2 a, PGA2 and PGB 2 • In this paper we report on the effect of applied archidonic acid, PGEt, gentlslC acid, acetylsalicylic acid, salicylic acid and oleic acid, on flowering of aseptic excised Z. Pflanzenphysial. Bd. 88. S. 423-429. 1978.
424
E. G. GROENEWALD and ]. H. VISSER
Pharbitis shoot apices kept under short day conditions (8 hr light - 16 hr darkness). The concentration of the compounds was 10- 4 M. Also, on the effect of PGE h PGE 2 , PGF 1 a, PGF 2a, PGA h PGA 2, PGB 1 and PGB 2 and arachidonic acid on flowering of aseptic excised Pharbitis shoot apices kept under long days (8 hr darkness - 16 hr light). The concentrations of the compounds were 10- 4 M, 10- 5 M and 10-6 M. Material and Methods Pharbitis nil Chois. Strain «Violet » seeds were treated with conc. H 2S0 4 for 45 min., rinsed with dist. H 20 and sterilized in 70 % EtOH for 10 min., followed by 60 min. in 10 Ofo Ca(OClh and finally in a mixture of 0.1 Ofo HgCl 2 and 0.1 Ofo sodium lauryl sulphate for 10 min. The sterilized seeds were immediately rinsed in 4 changes of sterile dist. H 20. The sterilized seeds were placed onto wet cotton wool contained in previously sterilized 250 ml erlenmeyer flasks. The flasks containing the seeds were kept for 3 days in a constant tempo room (27 °C) and under continuous light (3450 lux) supplied by cool white fluorescent tubes. At the end of this period the Pharbitis seedlings were about 4 cm high, and using aseptic techniques the hypocotyls were cut 2 cm below the growing points and the cotyledons were removed. The plant segments obtained in this way were sterilized for 5 min. in 5 % Ca(OClh and rinsed with sterile dist. H 20. This second sterilization treatment was necessary because of a fungus that was hard to get rid of and was probably present inside the testa of the seed. The sterilized plant segments were trimmed at the cut edges and placed upright in 5 ml agar nutrient medium contained in test tubes (23 mm X 195 mm). The composition of the nu trient medium was mineral salts (LINSMAIER and SKOOG, 1965), sucrose 3
Fig. 1: A Pharbitis shoot segment (apex) in nutrient agar medium in a test tube.
z.
Pjlanzenphysiol. Bd. 88. S. 423-429. 1978.
The effect of arachidonic acid
425
ml of a sodium carbonate solution (0.2 mg/mI H 20). The pH was measured with pHydrion paper and adjusted to between 6 and 7.5. The dissolved compound was then filtered through a Millipore filter (0.45 ftm pore size). The filter sterilized dissolved compound (0.2 ml!5 ml agar medium) was added to the autoclaved agar nutrient medium when it reached a tempo of 40°-45 oe and it was weIl mixed and aIlowed to set. The shoot apices were usuaIly kept in nutrient ag ar medium without any of the test compounds added, for 3 days, to make certain that the apices were not infected with fungus. The apices were kept at 25 oe and under long day conditions (8 hr darkness - 16 hr light). At the end of this period a smaIl segment was cut off (ca. 5 mm) from the cut end of the hypocotyl of each plant segment (apex) and the apices were transferred to agar nutrient medium containing the test compound (Fig. 1). The test tubes containing the shoot apices were kept at 25 oe and either under long day conditions (8 hr darkness - 16 hr light) or short day conditions (8 hr light - 16 hr darkness). The light intensity was 8600 lux and was supplied by fluorescent tubes and incandescent lamps.
Results and Discussion
In the experiment where arachidonic acid, PGEt, gentisic acid, ac'etylsalicylic acid, salicylic acid and oleic acid were applied to excised shoot apices under inductive conditions (short days) it was found that arachidonic acid and PGE t hastened flower formation by 29 days and 28 days respectively as compared with controls. A Pharbitis plantlet with a terminal flower, in a test tube, is depic'ted in Fig. 2. The benzoic acid derivatives (gentisic acid, acetylsalicylic acid and salicylic acid) a11 inhibited flowering completely. The results are summarized in Table 1.
Fig. 2: A Pharbitis plantlet which developed from an apex in a test tube. Note the flower. Z. Pflanzenphysiol. Bd. 88. S. 423-429. 1978.
426
E. G. GROENEWALD and
J.
H. VISSER
Table 1: EHect of various chemical compounds (10-4 M) on flowering of excised shoot apices of Pharbitis kept under inductive (8 hr light - 16 hr darkness) conditions. Five replicates were used per treatment. Treatment
Number of plants that flowered
Flowering first noticed (days after start of experiments)
Control Arachidonic acid PGE 1 Gentisic acid Acetylsalicylic acid Salicylic acid Oleic acid
5 5 3
45 16 17
o o
o
o
In the experiment where arachidonic acid, PGEt, PGE 2, PGF1a, PGF 2a, PGAt, PGA 2 , PGB 1 and PGB 2 were applied to excised apices under non-inductive (long day) conditions it was found that none of the treatments induced flower formation. However, in most of the treatments the higher concentrations (10- 4 M and 10-5 M) caused a reduction in leaf bl ade growth of the plantlets. Three types of responses were encountered, namely, A, Band C and they are depicted in Fig. 3, 4 and 5 respectively. The results are summarized in Table 2.
Fig.3
Fig.4
Fig.5
Fig. 3: EHect of prostagiandin (10-4 M) on the growth of a shoot apex of Pharbitis under long day conditions. Leaf growth was drastically reduced (see text for details). Fig. 4: EHect of prostagiandin (10-5 M) on the growth of a shoot apex of Pharbitis under long day conditions. The shape of the leaf blade was altered by the treatment (arrowhead shape) (see text for details). Fig. 5: EHect of prostagiandin (10- 6 M) on the growth of a shoot apex of Pharbitis under long day conditions. Plantlet similar to untreated (control) apex (see text for details). Z. Pjlanzenphysiol. Bd. 88. S. 423-429. 1978.
The effect of arachidonic acid
427
Table 2: Summary of results obtained with the addition of arachidonic acid and different prostaglandins to the nutrient agar in which shoot apices of Pharbitis were grown under long days (8 hr darkness - 16 hr light). Responses to the added substances are designated A, Band C and are depicted by Figs. 3, 4 and 5. The fractions indicate the number of apices out of the total of replicates that gave a certain response. Controls gave response C.
Treatment
Reaction to different concentrations 10-sM 10-4M 10-6 M
Arachidonic acid PGE1 PGE 2 PGF 1a PGF 2a PGA 1 PGA2 PGB 1 PGB 2
A 4/S A 2/r. A 2/S B 4/S A 3/S A 2/ S A 1/2 B 4/4 B 3/.
B B B B B B B B B
3/S 4/. 3/S 3/r. 4/S 31" 3/4 3/3 4/4
C C C B C C C C C
3/3
s/s s/s 1,s /s
4/r. 4/. 5/S 4/4
It is clear from the results obtained in the short day experiment that both arachidonic acid and PGE1 hastened flowering. Arachidonic acid is aprecursor for PGE2 and PGF2 a. The benzoic acid derivatives, namely, gentisic acid, acetylsalicylic acid and salicylic acid are all inhibitors of prostagiandin synthesis. Of the three acids gentisic acid is the most potent inhibitor of prostagiandin synthetase followed by acetylsalicylic acid and salicylic acid (FLOWER, 1974). Another benzoic acid derivative namely, gallic acid was reported (PRYCE, 1972) to be a natural inhibitor of flowering in the short day plant Kalanchoe blossfeldiana. It was, however, found by CLELAND and A]AMI (1974) that honeydew produced by an aphid when feeding on flowering or vegetative plants of the short day plant Xanthium strumarium produced an active substance capable of inducing flowering in the long day plant Lemna gibba G3. The ac'tive substance was found to be salicylic acid. This is in contrast to our findings with Pharbitis nil. Oleic acid, a fatty acid, is also an inhibitor of prostagiandin synthesis (PACE-AsCIAK and WOLFE, 1968). The nature of inhibition is probably competitive. In the experiments conducted under long day conditions the Pharbitis plantlets could not be induced to flower. Since the plantlets produced leaves it is possible that production of natural inhibitors of flowering could have been produced thus preventing flowering. In this regard DE FOSSARD (1974) advocates that specimens should be grown under inductive conditions in order to avoid natural inhibitor formation. Specimens should also be defoliated in order to reduce the quantity of natural florigen produc'tion under the conditions of the test and further specimens should be reduced to the tissue which responds to natural florigens, i.e. the shoot apical meristem in order to avoid indirect responses of applied substances. On the evidence obtained thus far one can put forward a hypothetical scheme for the control of flowering by prostaglandins and phenolic acids as depicted in Fig. 6.
z.
Pjlanzenphysiol. Bd. 88. S. 423-429. 1978.
428
E. G. GROENEWALD and
J.
H. VISSER
Pathways 01 biosynthesis ot prostaglandins and a few ph.nolic acids which may play a rol. in flowaring of Pharbitis.
GIUrSe
Phospholipids
1
Phospholipase H
~
C-20 unsaturated Fatty acids H H H Arachidonic acid Shikimic aci 8,11.14-Eicosatrienoic acid 5.8.11.14,17'Eicosapentaenoic acid
Qc~~-COOH
PG synthetase iCiH2 I-Phenylalanine
r PAL
Prostaglandins
1P
Flowering
Inhibitors ar PGsynthetase and Flowerlng o-Äcetylsallcyllc acid Sallcyllc acid Gentlslc acid Pt1enylbutazone Indomethacln Nfflumlcacld
ry-~-COOH t-Cinnamlc acid
\
,/
(}
'=&
7
~OOH Salicylic acid
Fig. 6: A hypothetical scheme depicting the possible interrelationship between prostaglandins and phenolic inhibitors (salicylic acid) on flowering of Pharbitis nil.
There is chromatographie evidence that salicylic acid (GROENEWALD and VISSER, unpublished results) amongst other phenolic acids, is produced in the cotyledons of Pharbitis nil and it is possible that salicylic acid could be a natural inhibitor of flowering in Pharbitis. In this regard it is interesting to note that phenolic acids arise from the action of phenyl alanine ammonia-lyase (PAL) and that light has a stimulatory effect on the enzyme, and thus the production of phenolic acids. It is thus possible that under long day conditions relatively large amounts of phenolic acids may be produced, as was found by PRYCE (1972) in the case of gallic acid, and this may be the cause of flower inhibition, sinc'e a number of phenolic acids are kno'wn inhibitors of prostagiandin synthetase (FLOWER, 1974). Acknowledgements We thank Dr. J. E. PIKE, Upjohn, Kalamazoo, Mich., U.S.A. for the prostaglandins used in this study. We also thank the C.S.I.R. for financial assistance and the Margaretha Mes Institute, University, of Pretoria, for the facilities provided. Z. Pjlanzenphysiol. Bd. 88. S. 423-429. 1978.
The effect of arachidonic acid
429
References ATTREP, K. A., J. M. MARIANI, Jr., and M. ATTREP, Jr.: Search for prostagiandin Al in onion. Lipids 8, 484-486 (1973). CLELAND, C. F. and A. AJAMI: Identification of the flower-inducing factor isolated from aphid honeydew as being salicylic acid. Plant Physiol. 54, 904-906 (1974). DE FOSSARD, R. A.: Flower initiation in tissue and organ cultures. In: H. E. STREET (Ed.). Tissue culture and Plant Science, pp. 193-212 Academic Press, London and New York, 1974. FLOWER, R. J.: Drugs which inhibit prostagiandin biosynthesis. Pharmacol. Rev. 26, (1), 33-67 (1974). GROENEWALD, E. G. and J. H. VISSER: The effect of certain inhibitors of prostagiandin biosynthesis on flowering of Pharbitis nil. Z. Pflanzenphysiol. 71, 67-70 (1974). LINsMAIER, ELFRIEDE, M and F. SKOOG: Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant 18, 100-127 (1965). MIYARES CAO, C. M. and E. MENENDEZ CEPERO: Identification of prostaglandinlike substances in plants. In: B. SAMUELSSON and R. PAOLETTI (Eds.) Advances in prostagiandin and thromboxane research p. 877. Vol. 2., Raven Press, New York, 1976. PACE-AsCIAK, C. and L. S. WOLFE: Inhibition of prostagiandin synthesis by oleic, linoleic and linolenic acids. Biochim. Biophys. Acta 152, 784-787 (1968). PRYCE, R. J.: Gallic acid as a natural inhibitor of flowering in Kalanchoe blossfeldiana. Phytochemistry 11, 1911-1918 (1972). E. G. GROENEWALD, Department of Botany, University of the O. F. S. Bloemfontein 9300, South Africa. J. H. VISSER, Department of Botany, University of Pretoria, Pretoria, 0002, South Africa.
z.
Pflanzenphysiol. Bd. 88. S. 423-429. 1978.