Planta (Berl.) 112, 87--90 (1973) 9 by Springer-Verlag 1973
Short Communication The Isolation of an Abscisic-acid Metabolite, 4'-Dihydrophaseic Acid, from Non-imbibed Phaseolus vulgaris Seed* D. C. W a l t o n , B. Dorn, a n d J. F e y Department of Chemistry, State University of New York, College of Environmental Science and Forestry, Syracuse, New York 13210, USA Received January 17 / February 28, 1973
Summary. Naturally occurring 4'-dihydrophaseic acid (DPA) has been isolated from mature, non-imbibed bean seed. The concentrations of abscisie acid (ABA), phaseie acid (PA) and DPA in the seed were estimated to be 0.06, 0.11 and 5.95 mg/kg dry wt., respectively. The results suggest that DPA is a major inactivation product of ABA in this tissue. The possible pathway from ABA to DPA is discussed. W h e n excised e m b r y o n i c axes f r o m Phaseolus vulgaris L., cv. W h i t e Marrowfat, are i n c u b a t e d w i t h 2 [I~CJABA, two m e t a b o l i t e s are r a p i d l y f o r m e d ( S o n d h e i m e r et al., 1971; W a l t o n a n d Sondheimer, 1972a). One m e t a b o l i t e (M-2) accumulates, while t h e o t h e r (M-l) r e m a i n s a t a rela t i v e l y low concentration. The m a j o r m e t a b o l i t e h a s r e c e n t l y been identified as D P A (Fig. 1) a n d t h e m i n o r m e t a b o l i t e as P A (Tinelli et al., 1973). P A has p r e v i o u s l y been i s o l a t e d f r o m Phaseotus coccineus L. seed (MeMillan a n d P r y c e , 1968), a n d r e p o r t e d to occur in c o t t o n f r u i t (Davis et al., 1972) a n d d r o u g h t - s t r e s s e d sugar cane (Most, 1971). I t has also been r e p o r t e d as a m e t a b o l i t e of 2114C]ABA in t o m a t o p l a n t s (Milborrow, 1972). D P A h a d n o t been r e p o r t e d prior to its c h a r a c t e r i z a t i o n as a n A B A m e t a b o l i t e in excised e m b r y o n i c b e a n axes. This r e p o r t describes t h e isolation of n a t u r a l l y occurring D P A f r o m m a t u r e noni m b i b e d Phaseolus vulgaris seed a n d c o m p a r e s t h e c o n c e n t r a t i o n s of A B A , P A a n d D P A in t h e seed. 2 kg of nonimbibed seed (Phaseolns vulgaris L. ev. White Marrowfat obtained from Agway, Canandaigua, N.Y., U.S.A.) were ground in a Wiley mill and the powder extracted overnight at 4 ~ with 12 1 of 80% methanol. The methanol was removed from the filtered extract in a rotary evaporator in vacuo at 35 ~ At this stage, approximately 300000 dpm each of 2114C]ABA, 2114C]PA, 2[I~C]DPA were added to the aqueous solution to act as markers and to enable us to estimate losses during purification. The quantities of ABA, PA and DPA added to the solution were approximately 2, 22 and 12 ~g, respectively. The radioactive PA and DPA had been isolated from bean seed previously incubated with 2114C]ABA (spec. act. 10 mCi/
* Abbreviations: ABA abscisic acid, DPA 4'-dihydrophaseic acid, PA phaseie acid.
88
D.C. Walton et al. :
H O ~ 3'
JCO2H
Pig. 1.4'-Dihydrophaseic acid mmol) (Sondheimer and Tinelli, 1971). The p i t of the aqueous solution was adjusted to 8 with sodium bicarbonate, and then extracted 3 times with equal volumes of ethyl acetate; the ethyl acetate was discarded. The aqueous solution was adjusted to p H 3 with HC1 and extracted 3 times with equal volumes of water-saturated nbutanol. The butanol extracts were combined and evaporated to dryness i n vacuo at 35 ~ The residue (5 g) was taken up in 140 ml methanol to which was added 700 ml of acetone. The resulting precipitate was removed by centrifugation and discarded. The supernatant was evaporated to dryness and the residue taken up in 100 ml water. The filtered solution was added to a 2 • 30 cm column of charcoal: celite (3:2) and eluted with a step gradient of 10, 20, 30, 40, and 60% acetone in water. Approximately 500 ml of each concentration of aqueous acetone were used in the elution. P A and DPA were eluted from the column with 40% acetone and the ABA with 60 %. The fractions containing radioactivity were combined, evaporated, and then streaked on 2 mm thick Brinckmann precoated Silica gel G thin-layer chromatography (TLC) plates with the appropriate standards. The plates were developed with chloroform: methanol: water (75: 22: 3, v/v), the radioactive bands located by autoradiography and eluted with ethanol: acetone (1 : 1, v/v). The eluted bands were rechromatogrammed on the same type of TLC plate, using benzene: ethyl acetate: acetic acid (50: 5: 2, v/v) as the developing solvent. The radioactive bands were located and eluted as described above. For quantification of ABA, the concentration of (q-)-ABA in the band eluted from the second TLC plate was determined by ORD using a Jasco Model ORD/UV 5 spectropolarimeter (Milborrow, 1968). ~or quantification of PA, the P A eluted from the second TLC plate was methylated with diazomethane (Schlenk and Gellerman, 1960) and chromatogrammed on 0.25-mm-thick Brinckmann precoated Silica-gel G TLC plates, using isooctane: ethyl acetate (1 : 1, v/v) as the developing solvent. The concentration of the eluted methyl phaseate was determined by gas-liquid chromatography (GLC) using a previously purified sample of methyl phaseate as a standard. GLC was done with a Hewlett-Packard Model 5750 instrument equipped with a tritium electroncapture detector. The system used a 183 cm • 2 mm glass column packed with 3% DC-200 on 60/80 mesh Gas-Chrom Q. The column temperature was 169 ~ and the carrier gas argon:methane (95:5) at a flow rate of 25 ml/min. Under these conditions the methyl phaseate had a retention time of approximately 11 min and it was possible to detect less than 1 rig. For quantification of DPA, the DPA eluted from the second TLC plate was methylated with diazomethane and chromatogrammed as described for methyl phaseate. The radioactive band was eluted and the methyl 4'-dihydrophaseate crystallized twice from ether. The concentration of methyl 4'-dihydrophaseate was estimated from its absorbance at 267 nm (e 19900) in methanol (Tinelli e t a l . , 1973). Losses of the three compounds during purification were estimated by liquid scintillation counting.
Isolation ol an Abscisic-acid Metabolite
89
Table 1. Concentrations of ABA, PA and DPA in mature non-imbibed bean seeds Estimates adjusted for losses during purification Compound
Final recovery of added radioactivity (%)
Concentration (mg/kg dry wt.)
ABA PA DPA
34 18 10
0.06 0.11 5.95
The concentrations of ABA, PA and D P A estimated to occur in mature non-imbibed Phaseolus vulgaris seed are compared in Table 1. These estimates include corrections for losses during purification as determined by loss of radioactivity from the marker compounds added at the beginning of the purification process. The crystalline methyl4'-dihydrophaseate obtained from the non-imbibed seed had m . p . , NMR, mass spectra and chemical reactivity comparable to those obtained with the compound isolated from excised embryonic axes which had been incubated with high concentrations of 2[I~C]ABA (Tinelli et al., 1973). The results shown in Table 1 suggest t h a t D P A is a naturally occurring metabolite of ABA in bean seeds and agree with the results obtained when either excised embryonic axes or intact seeds have been incubated with 2 [14C]ABA (Walton and Sondheimer, 1972 a; Sondheimer et al., 1971). Several other more polar metabolites are formed when unimbibed bean seeds are incubated with 2114C]ABA for 80 h, but D P A is still the major metabolite observed (unpublished results). Three pathways can be postulated for the conversion of ABA to DPA: (1) A B A - ~ 6'-hydroxy-ABA--> P A - ~ DPA. (2) ABA--~ 6'-hydroxy-ABA-~ 4'-dihydro,6'-hydroxy-ABA--> DPA. (3) A B A - ~ 4'-dihydro-ABA-~ 4'-dihydro,6'-hydroxy-ABA--> DPA. Of the various possible intermediates only 6'-hydroxy ABA and PA have been observed as metabolites of 2114C]ABA (Milborrow, 1971; Walton and Sondheimer, 1972a). We have not observed 6'-hydroxy-ABA, but part of the PA we observe m a y have been formed from 6'-hydroxyABA during extraction and purification, since the presence of the conjugated ketone makes non-enzymatic ring closure very favorable. Neither 4'-dihydro-ABA nor 4'-dihydro-6'-hydroxy-ABA contain a conjugated ketone so t h a t failure to observe them due to non-enzymatic ring closure during purification is unlikely. When either 2 [14C]PA or 2 [laC]4'-dihydroABA was fed to excised bean axes 2114C]DPA was formed (Walton and Sondheimer, 1972a, b). The conversion from PA was essentially complete but t h a t from 4'-dihydro-ABA was only very slight. ABA was also
90
D.C. Walton et al.: Isolation of an Abscisic-acid Me~abolite
f o r m e d f r o m t h e 4 ' - d i h y d r o - A B A so t h a t even t h e slight a m o u n t of D P A o b t a i n e d n e e d n o t h a v e b e e n f o r m e d b y p a t h w a y 3. F o r t h e a b o v e reasons we feel t h a t p a t h w a y 1 is t h e m o s t p r o b a b l e for t h e conversion of D P A f r o m A B A , a l t h o u g h we c a n n o t rule o u t t h e p o s s i b i l i t y of 2 a n d 3. I f p a t h w a y 1 is i n v o l v e d , t h e n P A is n o t m e r e l y a n a r t i f a c t of i s o l a t i o n as s u g g e s t e d b y M i l b o r r o w (1971). D P A h a s b e e n t e n t a t i v e l y i d e n t i f i e d as a m e t a b o l i t e of 2114C]ABA in ash seed (E. Sondheimer, p e r s o n a l c o m m u n i c a t i o n ) a n d in horsechestn u t buds. These results suggest t h a t D P A m a y be a n A B A m e t a b o l i t e in a v a r i e t y of p l a n t s , r a t h e r t h a n b e i n g r e s t r i c t e d t o Phaseolus. This work was supported by National Science Foundation grant GB 29428. We are grateful to Dr. E. Sondheimer, State University of New York College of Environmental Science and Forestry, Syracuse, for 2[laC]ABA and to E. Tinelli for technical assistance.
References Davis, L. A., Lyon, J. A., Addicott, F. T. : Phaseie acid: Occurrence in cotton fruit; acceleration of abscission. Planta (Berl.) 102, 294-301 (1972). MacMillan, J., Pryce, R. J.: Phaseic acid, a putative relative of abscisie acid, from seed of Phaseolus multi/lotus. Chem. Comm. 1968, 124-126. Milborrow, B. V. : Identification and measurement of (~)-abseisic acid in plants. In: Biochemistry and physiology of plant growth substances, p. 1531-1545, Wightman, F., Setterfield, G., eds. Ottawa: Runge Press 1968. Milborrow, B. V.: Abscisic acid. In: Aspects of terpenoid chemistry and biochemistry, p. 137-151, Goodwin, T. W., ed. New York: Acad. Press 1971. Milborrow, B. V.: The biosynthesis and degradation of abscisic acid. In: Plant growth substances, p. 281-290, Cart, D. J., ed. Berlin-Heidelberg-New York: Springer 1972. Most, B. H.: Abscisic acid in immature apical tissue of sugar cane and in leaves of plants subjected to drought. P1anta (Berl.) 101, 67-75 (1971). Schlenk, H., Gellerman, J. L.: Esterification of fatty acids with diazomethane on a small scale. Analyt. Chem. 82, 1412-1414 (1960). Sondheimer, E., Galson, E. C., Chang, Y. P., Walton, D. C.: Asymmetry, its importance to the action and metabolism of abscisie acid. Science 174, 829-831 (1971). Sondheimer, E., Tinelli, E . T . : Improved synthesis of (RS)-2-14C-abscisic acid. Phytochem. 19, 1663-1664 (1971). Tinelli, E.T., Sondheimer, E., Walton, D.C., Gaskin, P., McMillan, J.: Metabolites of 2-14C-abscisie acid. Tetrahedron Letters 1978, 139-140. Walton, D. C., Sondheimer, E.: Metabolism of 2-14C-(:J: )-abscisic acid in excised bean axes. Plant Physiol. 49, 285-289 (1972a). Walton, D. C., Sondheimer, E. : Activity and metabolism of 14C-(• )-abscisic acid derivatives. Plant Physiol. 49, 290-292 (1972).
Short Communication The Isolation of an Abscisic-acid Metabolite, 4'-Dihydrophaseic Acid, from Non-imbibed Phaseolus vulgaris Seed* D. C. W a l t o n , B. Dorn, a n d J. F e y Department of Chemistry, State University of New York, College of Environmental Science and Forestry, Syracuse, New York 13210, USA Received January 17 / February 28, 1973
Summary. Naturally occurring 4'-dihydrophaseic acid (DPA) has been isolated from mature, non-imbibed bean seed. The concentrations of abscisie acid (ABA), phaseie acid (PA) and DPA in the seed were estimated to be 0.06, 0.11 and 5.95 mg/kg dry wt., respectively. The results suggest that DPA is a major inactivation product of ABA in this tissue. The possible pathway from ABA to DPA is discussed. W h e n excised e m b r y o n i c axes f r o m Phaseolus vulgaris L., cv. W h i t e Marrowfat, are i n c u b a t e d w i t h 2 [I~CJABA, two m e t a b o l i t e s are r a p i d l y f o r m e d ( S o n d h e i m e r et al., 1971; W a l t o n a n d Sondheimer, 1972a). One m e t a b o l i t e (M-2) accumulates, while t h e o t h e r (M-l) r e m a i n s a t a rela t i v e l y low concentration. The m a j o r m e t a b o l i t e h a s r e c e n t l y been identified as D P A (Fig. 1) a n d t h e m i n o r m e t a b o l i t e as P A (Tinelli et al., 1973). P A has p r e v i o u s l y been i s o l a t e d f r o m Phaseotus coccineus L. seed (MeMillan a n d P r y c e , 1968), a n d r e p o r t e d to occur in c o t t o n f r u i t (Davis et al., 1972) a n d d r o u g h t - s t r e s s e d sugar cane (Most, 1971). I t has also been r e p o r t e d as a m e t a b o l i t e of 2114C]ABA in t o m a t o p l a n t s (Milborrow, 1972). D P A h a d n o t been r e p o r t e d prior to its c h a r a c t e r i z a t i o n as a n A B A m e t a b o l i t e in excised e m b r y o n i c b e a n axes. This r e p o r t describes t h e isolation of n a t u r a l l y occurring D P A f r o m m a t u r e noni m b i b e d Phaseolus vulgaris seed a n d c o m p a r e s t h e c o n c e n t r a t i o n s of A B A , P A a n d D P A in t h e seed. 2 kg of nonimbibed seed (Phaseolns vulgaris L. ev. White Marrowfat obtained from Agway, Canandaigua, N.Y., U.S.A.) were ground in a Wiley mill and the powder extracted overnight at 4 ~ with 12 1 of 80% methanol. The methanol was removed from the filtered extract in a rotary evaporator in vacuo at 35 ~ At this stage, approximately 300000 dpm each of 2114C]ABA, 2114C]PA, 2[I~C]DPA were added to the aqueous solution to act as markers and to enable us to estimate losses during purification. The quantities of ABA, PA and DPA added to the solution were approximately 2, 22 and 12 ~g, respectively. The radioactive PA and DPA had been isolated from bean seed previously incubated with 2114C]ABA (spec. act. 10 mCi/
* Abbreviations: ABA abscisic acid, DPA 4'-dihydrophaseic acid, PA phaseie acid.
88
D.C. Walton et al. :
H O ~ 3'
JCO2H
Pig. 1.4'-Dihydrophaseic acid mmol) (Sondheimer and Tinelli, 1971). The p i t of the aqueous solution was adjusted to 8 with sodium bicarbonate, and then extracted 3 times with equal volumes of ethyl acetate; the ethyl acetate was discarded. The aqueous solution was adjusted to p H 3 with HC1 and extracted 3 times with equal volumes of water-saturated nbutanol. The butanol extracts were combined and evaporated to dryness i n vacuo at 35 ~ The residue (5 g) was taken up in 140 ml methanol to which was added 700 ml of acetone. The resulting precipitate was removed by centrifugation and discarded. The supernatant was evaporated to dryness and the residue taken up in 100 ml water. The filtered solution was added to a 2 • 30 cm column of charcoal: celite (3:2) and eluted with a step gradient of 10, 20, 30, 40, and 60% acetone in water. Approximately 500 ml of each concentration of aqueous acetone were used in the elution. P A and DPA were eluted from the column with 40% acetone and the ABA with 60 %. The fractions containing radioactivity were combined, evaporated, and then streaked on 2 mm thick Brinckmann precoated Silica gel G thin-layer chromatography (TLC) plates with the appropriate standards. The plates were developed with chloroform: methanol: water (75: 22: 3, v/v), the radioactive bands located by autoradiography and eluted with ethanol: acetone (1 : 1, v/v). The eluted bands were rechromatogrammed on the same type of TLC plate, using benzene: ethyl acetate: acetic acid (50: 5: 2, v/v) as the developing solvent. The radioactive bands were located and eluted as described above. For quantification of ABA, the concentration of (q-)-ABA in the band eluted from the second TLC plate was determined by ORD using a Jasco Model ORD/UV 5 spectropolarimeter (Milborrow, 1968). ~or quantification of PA, the P A eluted from the second TLC plate was methylated with diazomethane (Schlenk and Gellerman, 1960) and chromatogrammed on 0.25-mm-thick Brinckmann precoated Silica-gel G TLC plates, using isooctane: ethyl acetate (1 : 1, v/v) as the developing solvent. The concentration of the eluted methyl phaseate was determined by gas-liquid chromatography (GLC) using a previously purified sample of methyl phaseate as a standard. GLC was done with a Hewlett-Packard Model 5750 instrument equipped with a tritium electroncapture detector. The system used a 183 cm • 2 mm glass column packed with 3% DC-200 on 60/80 mesh Gas-Chrom Q. The column temperature was 169 ~ and the carrier gas argon:methane (95:5) at a flow rate of 25 ml/min. Under these conditions the methyl phaseate had a retention time of approximately 11 min and it was possible to detect less than 1 rig. For quantification of DPA, the DPA eluted from the second TLC plate was methylated with diazomethane and chromatogrammed as described for methyl phaseate. The radioactive band was eluted and the methyl 4'-dihydrophaseate crystallized twice from ether. The concentration of methyl 4'-dihydrophaseate was estimated from its absorbance at 267 nm (e 19900) in methanol (Tinelli e t a l . , 1973). Losses of the three compounds during purification were estimated by liquid scintillation counting.
Isolation ol an Abscisic-acid Metabolite
89
Table 1. Concentrations of ABA, PA and DPA in mature non-imbibed bean seeds Estimates adjusted for losses during purification Compound
Final recovery of added radioactivity (%)
Concentration (mg/kg dry wt.)
ABA PA DPA
34 18 10
0.06 0.11 5.95
The concentrations of ABA, PA and D P A estimated to occur in mature non-imbibed Phaseolus vulgaris seed are compared in Table 1. These estimates include corrections for losses during purification as determined by loss of radioactivity from the marker compounds added at the beginning of the purification process. The crystalline methyl4'-dihydrophaseate obtained from the non-imbibed seed had m . p . , NMR, mass spectra and chemical reactivity comparable to those obtained with the compound isolated from excised embryonic axes which had been incubated with high concentrations of 2[I~C]ABA (Tinelli et al., 1973). The results shown in Table 1 suggest t h a t D P A is a naturally occurring metabolite of ABA in bean seeds and agree with the results obtained when either excised embryonic axes or intact seeds have been incubated with 2 [14C]ABA (Walton and Sondheimer, 1972 a; Sondheimer et al., 1971). Several other more polar metabolites are formed when unimbibed bean seeds are incubated with 2114C]ABA for 80 h, but D P A is still the major metabolite observed (unpublished results). Three pathways can be postulated for the conversion of ABA to DPA: (1) A B A - ~ 6'-hydroxy-ABA--> P A - ~ DPA. (2) ABA--~ 6'-hydroxy-ABA-~ 4'-dihydro,6'-hydroxy-ABA--> DPA. (3) A B A - ~ 4'-dihydro-ABA-~ 4'-dihydro,6'-hydroxy-ABA--> DPA. Of the various possible intermediates only 6'-hydroxy ABA and PA have been observed as metabolites of 2114C]ABA (Milborrow, 1971; Walton and Sondheimer, 1972a). We have not observed 6'-hydroxy-ABA, but part of the PA we observe m a y have been formed from 6'-hydroxyABA during extraction and purification, since the presence of the conjugated ketone makes non-enzymatic ring closure very favorable. Neither 4'-dihydro-ABA nor 4'-dihydro-6'-hydroxy-ABA contain a conjugated ketone so t h a t failure to observe them due to non-enzymatic ring closure during purification is unlikely. When either 2 [14C]PA or 2 [laC]4'-dihydroABA was fed to excised bean axes 2114C]DPA was formed (Walton and Sondheimer, 1972a, b). The conversion from PA was essentially complete but t h a t from 4'-dihydro-ABA was only very slight. ABA was also
90
D.C. Walton et al.: Isolation of an Abscisic-acid Me~abolite
f o r m e d f r o m t h e 4 ' - d i h y d r o - A B A so t h a t even t h e slight a m o u n t of D P A o b t a i n e d n e e d n o t h a v e b e e n f o r m e d b y p a t h w a y 3. F o r t h e a b o v e reasons we feel t h a t p a t h w a y 1 is t h e m o s t p r o b a b l e for t h e conversion of D P A f r o m A B A , a l t h o u g h we c a n n o t rule o u t t h e p o s s i b i l i t y of 2 a n d 3. I f p a t h w a y 1 is i n v o l v e d , t h e n P A is n o t m e r e l y a n a r t i f a c t of i s o l a t i o n as s u g g e s t e d b y M i l b o r r o w (1971). D P A h a s b e e n t e n t a t i v e l y i d e n t i f i e d as a m e t a b o l i t e of 2114C]ABA in ash seed (E. Sondheimer, p e r s o n a l c o m m u n i c a t i o n ) a n d in horsechestn u t buds. These results suggest t h a t D P A m a y be a n A B A m e t a b o l i t e in a v a r i e t y of p l a n t s , r a t h e r t h a n b e i n g r e s t r i c t e d t o Phaseolus. This work was supported by National Science Foundation grant GB 29428. We are grateful to Dr. E. Sondheimer, State University of New York College of Environmental Science and Forestry, Syracuse, for 2[laC]ABA and to E. Tinelli for technical assistance.
References Davis, L. A., Lyon, J. A., Addicott, F. T. : Phaseie acid: Occurrence in cotton fruit; acceleration of abscission. Planta (Berl.) 102, 294-301 (1972). MacMillan, J., Pryce, R. J.: Phaseic acid, a putative relative of abscisie acid, from seed of Phaseolus multi/lotus. Chem. Comm. 1968, 124-126. Milborrow, B. V. : Identification and measurement of (~)-abseisic acid in plants. In: Biochemistry and physiology of plant growth substances, p. 1531-1545, Wightman, F., Setterfield, G., eds. Ottawa: Runge Press 1968. Milborrow, B. V.: Abscisic acid. In: Aspects of terpenoid chemistry and biochemistry, p. 137-151, Goodwin, T. W., ed. New York: Acad. Press 1971. Milborrow, B. V.: The biosynthesis and degradation of abscisic acid. In: Plant growth substances, p. 281-290, Cart, D. J., ed. Berlin-Heidelberg-New York: Springer 1972. Most, B. H.: Abscisic acid in immature apical tissue of sugar cane and in leaves of plants subjected to drought. P1anta (Berl.) 101, 67-75 (1971). Schlenk, H., Gellerman, J. L.: Esterification of fatty acids with diazomethane on a small scale. Analyt. Chem. 82, 1412-1414 (1960). Sondheimer, E., Galson, E. C., Chang, Y. P., Walton, D. C.: Asymmetry, its importance to the action and metabolism of abscisie acid. Science 174, 829-831 (1971). Sondheimer, E., Tinelli, E . T . : Improved synthesis of (RS)-2-14C-abscisic acid. Phytochem. 19, 1663-1664 (1971). Tinelli, E.T., Sondheimer, E., Walton, D.C., Gaskin, P., McMillan, J.: Metabolites of 2-14C-abscisie acid. Tetrahedron Letters 1978, 139-140. Walton, D. C., Sondheimer, E.: Metabolism of 2-14C-(:J: )-abscisic acid in excised bean axes. Plant Physiol. 49, 285-289 (1972a). Walton, D. C., Sondheimer, E. : Activity and metabolism of 14C-(• )-abscisic acid derivatives. Plant Physiol. 49, 290-292 (1972).
