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Cytokinins and Ancymidol inhibit Abscisic Acid Biosynthesis in Persea gratissima
Cytokinins and Ancymidol inhibit Abscisic Acid Biosynthesis in Persea gratissima A. K. COWAN and I. D. RAILTON Plant Growth Laboratory, Department of Plant Sciences, Rhodes University, Grahamstown 6140 South Africa Received January 23, 1987 . Accepted February 24, 1987
Summary The cytokinins, benzyl adenine, kinetin, isopentenyl adenine, and zeatin and the cytokinin
analogue, ancymidol all inhibited the biosynthesis of ahscisic acid from mevalonic acid in mesocarp tissue from ripe fruits of avocado {Persea gratissima}.
Key words: Abscisic acid biosynthesis. cytokinim, ancymidol, inhibition, avocado mesocarp.
Introduction Studies on the production of abscisic acid (ABA) by the fungus Cercospora rosicola, have shown that several cytokinins are potent inhibitors of the biosynthetic pathway to this important sesquiterpenoid plant growth hormone (Norman et aI., 1983 a). Investigations with [l-"C}farnesyl pyrophosphate ([j-1'}FPP) showed thaI one cytokinin, benzyl adenine, (BA), inhibited specifically, post-FPP steps in ABA biosynthesis in this fungus (Norman et a1., 1983 a). In other studies, it was demonstrated that several plant growth retardants and inhibitors of gibberellin (GA) biosynthesis, including ancymidol, which bears structural similarities to cytokinins, would also inhibit ABA production in C. rosicola (Norman et a1., 1983 b). The effect of these compounds on ABA biosynthesis in higher plant tissues has never been investigated. We therefore examined several cytokinins and the cytokinin analogue, ancymidol, as potential inhibitors of ABA biosynthesis in the mesocarp of fruits of Persea gratissima, the only well-documented higher plant system which will synthesize routinely, ABA from radioactive terpenoid precursors such as mevalonic acid (MV A) (Noddle and Robinson, 1969; Milborrow and Robinson, 1973; Cowan and RailIon, 1986). Materials and Methods Plant material Soft, ripe fruits of avocado (Persea gratissima) were purchased from a local trader and used immediately.
Application of compouTJdJ
Fruits were cut in half, de-stoned and the skin removed. Excised blocks of mesocarp (each ZOg) were pre-treated with either ancymidoI (SOO~) or the cytokinins BA, kinetin(K), zeaj. Piant Physinl. Vol. 130. pp. 213-217 (1987)
274
A. K.
COWAN
and I. D.
RAILTON
tin(Z) and isopentenyl adenine(IPA) or adenine (all 500 JLM), each dissolved in Tween 20: acetone :HzO (1: 1: 8, v/v), by infiltration via a series of cuts in the surface of the tissue. A total volume of 0.6 rul of each solution was administered in this way and the tissue blocks were left for 6 hours in continuous illumination (5500 lux) at 25°C in a humid environment under a large inverted crystallizing dish containing wet paper towels.
Biosynthesis 0/ABA This was carried out as described by Milborrow and Robinson (1973). R-{2- '4C}Mevalonolactone (R{2)4C}MVAL), (9.0kBq; sp. act. 1.9 GBq/mmol), purchased from Amersham International, UK, and disssolved in Tween 20: acetone: HzO (1: 1; 8, v/v) (0.2 ml), was administered to each block of avocado mesocarp which had been pre-treated with either ancymidol or each of the cytokinins or adenine for 6 hours. The blocks of mesocarp were then left under the same conditions for a further 24 hours prior to extraction. Extraction and purification ofABA
Tissues were homogenized in ice-cold ethyl acetate: methanol (50: SO, v/v). containing the antioxidant, butylated hydroxy toluene (BHT) (20 rugll), using an Ultraturrax top-drive blender. Each extract was left at 2 °C for 12 hours and was then filtered through Whatman No1 filter paper in a Buchner funnel. The residue was further extracted (2 x) and the combined fil· trates reduced in vacuo at 35°C to a small aqueous volume to which was added 100 m! of 0.5 M KzHP0 4 (pH 8.0). This was partitioned against EtzO (6 x) to remove neutral-basic impurities and then the aqueous phase was re.adjusted to pH 2.5 and further partitioned (6 x) against EtzO to extract the radioactive acids. The EtzO-soluble acid fraction was reduced to dryness in vacuo at 3S oC, dissolved in l.Oml methanol, K,HPO,·IQI,PO, buffer (20mM, pH 8.0) (32,68, v/v), loaded onto a Sep-pak C I8 cartridge (Waters Associates, Milford, MA. USA) and eluted with 6.0 ml of the same solution. The eluate was reduced in vacuo at 35°C and the residue dissolved in a small volume of ethyl acetate: methanol (50: 50, v/ v) for chromatography. Samples were separated by thin-layer chromatography (TIC) on silica gel GF 254 developed (2 x) to 15 em using the solvent system toluene: ethyl acetate; acetic acid (50: 30: 4, v/v) (Zeevaan and Milborrow, 1976). Plates were divided into 30 strips, each strip eluted with methanol and counted in a Beckmann LS5801 scintillation spectrometer using a cocktail of 2,5 diphenyloxazole in toluene (5 gil). Material co-chromatographing with authentic ABA was eluted with HzO-saturated ethyl acetate for further identification.
Characterization ofABA Putative ABA. newly biosynthcsized from R{2- 14C}MVAL was initially identified using the criteria of Milborrow and Robinson (1973) in which label co-migrating with ABA methyl ester (ABAMe) and retained in equal amounts in the diols of ABAMe following reduction of ABAMe with NaBH4. established the radioactive product as ABA. The product. as its methyl ester, was further characterized by radio-gas liquid chromatography (R-GLC). This was carried out on a dual column Perkin-Elmer 990 gas chromatograph interfaced with a Panax Radiogas Detector System as described before (Railton. 1980). Esterified samples were analysed on two separate liquid stationary phases. 2% 5£-30 and 1 % XE-60 on Ga.'ichrom Q (80-100 mesh) which were packed in silanizcd glass columns (1.8 m X 2 mm i.d.) with Ar as carrier gas at a flow rate of 43 ml/min and an oven temperature of 180°C. The effluent carrier gas was split between the flame ionization detector and gas flow proportional counter in a ratio of 1 : 15 and the [ 14C] compound was oxidized to t4C02 over copper oxide at 650°C in a silica furnace tube prior to entry into the proportional counter. The putative, radioactive ABAMe chromatographed as a single peak on both liquid stationary phases and exhib--ited identical retention times to those of authentic ABAMe viz: 2 % SE·30 (Rt - 3.5 min) and 1 % XE-60 (Rt - 3.7 min). Its identity as ABA was further established by u.v. isomerization of its methyl ester followed by analysis on R-GLC as above, when an additional radio.peak with
J. Plant Physiol. VoL 130. pp.
273-277 (1987)
Inhibition of Abscisic Acid Biosynthesis
275
identical retention times to those of t-ABAMe (2% SE-30, Rt == 4.1 min; 1 % XE-60, Rt == 4.7 min) was observed.
Results and Discussion The kinetics of ABA biosynthesis from R{2-"C}MVAL showed that maximum incorporation of label into ABA occurred 24 hours after supplying substrate to mesocarp tissue (Fig. 1). Thereafter, a decline in incorporation into ABA, which was accompanied by the appearance of label in phaseic acid and in the 1',4'-trans diol of ABA (Cowan and Railton, unpublished), probably resulted from ABA catabolism. Thus, in all subsequent feeds, metabolism was allowed to proceed for 24 hours .
•
z
2
"\.
~
o
I>
8z
\.
i
Ii
fig. 1: Kinetics of ABA biosynthesis from R{2_14C]MVAL by excised blocks (20g) of mesocarp from ripe fruits of avocado.
20
40
60
BO
100
TIME (hrs)
Table 1: The effect of cytOkinins and ancymidol on ABA biosynthesis by mesocarp tissue from ripe avocado fruits. Excised mesocarp (20 g) was pre-treated with either BA,K,Z,IPA,adenine or ancymidol (all 500~) for 6 hours prior to the application of R{2- 14C}MVAL (9.0 kBq). Metabolism of substrate was allowed to proceed for 24 hours and biosynthesized ABA was then isolated and characterized as described in «Matcials and Methods,.. All values were corrected for recovery. Treatment
Control Adenine
8A
K
IPA Z
Ancymidol
(Bq)
[ 14 C) Incorporated
(%)
% Inhibition
80.56 78.74 9.83 11.30 13.14 23.42 15.77
0.089 0.087 0.010 0.012 0.014 0.026 0.017
0.00 2.27 87.81 85.97
83.69 70.94 80.43
J. Plant Phy,iol. Vol.
130. pp. 273-277 (1987)
276
A. K. COWAN and I. D. RAILTON
The effect of cytokinins on ABA biosynthesis by avocado mesocarp is shown in Table 1. Whereas the parent molecule, adenine, was essentially inactive, all the N6
substituted purines, which normally exhibit high biological activity in other plant systems (Letham, 1978), markedly inhibited ABA biosynthesis in this tissue. All were supplied at 500 pM which has been reported to inhibit fungal ABA biosynthesis by 80-90% (Norman et al., 1983 a). As in the fungal studies, BA was the most inhibitory to ABA biosynthesis in avocado mesocarp, reducing incorporation of .label by almost 88 %. However, both IPA and K exhibited similar inhibitory activity whereas Z was the least active compound tested. The cytokinin analogue, ancymidol, also supplied at 500 I'M, inhibited ABA biosynthesis markedly in avocado mesocarp tissue
(Table 1). This is the first report that cytokinins and ancymidol can inhibit ABA biosynthesis in higher plant tissues. The steps in ABA biosynthesis in avocado mesocarp which are inhibited by these compounds is currently unknown. Coolbaugh (1984) has shown that both cytokinins and ancymidol can interact with cytochrome P450-mixed function oxidases in GA biosynthesis to inhibit kaurene oxidation in plants. This suggests that these compounds might also affect similarily catalyzed post-FPP oxidations in
fungal and plant ABA biosynthesis. However, studies on fungal ABA biosynthesis have shown that whereas BA does interfere with post-FPP steps (Norman et al., 1983 a), ancymidol appears to inhibit reactions which occur prior to FPP in the terpenoid pathway in this organism (Norman et al., 1986). Likewise, in cell-free extracts of Marah oreganus and shoot tips of Pisum sativum, ancymidol at higher concentrations than those required to inhibit kaurene oxidation, will also inhibit the conversion of
MVA to kaurene which includes several non-oxidative steps common to both ABA and kaurene biosynthesis (Coolbaugh and Hamilton, 1976). Whether or not these compounds exert the same inhibitory effect in all higher plant tissues remains to be investigated. Application of BA to excised barley leaves, for example, did not prevent wilt-induced increases in endogenous ABA levels (Stewart et aI., 1986), a phenomenon believed to involve elevated rates of ABA biosynthesis from MVA (Milborrow and Noddle, 1970). However, recent evidence (Railton and Cowan, 1987) suggests that water stress operates in barley leaves to reduce the catabolism of ABA rather than to increase its biosynthesis which might help explain these apparently anomalous findings. Acknowledgements This work was supported by grants from the CSIR(FRD) Pretoria South Africa to I.D, Railton.
References
R. c.: Inhibition of ent-kaurene oxidation by cytokinins.]. Plant Growth Regul. 3,97 -109 (1984). COOLBAUGH, R. C. and R. HAMILTON: Inhibition of ent-kaurene oxidation and growth by a-cyclopropyl-a[p-methoxyphenyl]-5-pyrimidine methyl alcohol. Plant Physiol. 57, 245-248 (1976). COOLBAUGH,
f Plant PIry,ioi. Vol. 130. pp.
213 - 217 (1987)
Inhibition of Abscisic Acid Biosynthesis
277
COWAN, A. K. and L D. RAILTON: Chloroplasts and the biosynthesis and catabolism of abscisic acid_ J. Plant Growth Regul. 4, 211- 224 (1986)_
lETIiAM, D. S.: Cytokinins. In: LETHAM, D. S., P. B. GOODWIN, T.]. V. HIGGINS (Eds.): Phytohormones and related compounds: Acomprehensive treatise. Elsevier/North Holland Biomedical Press, Amsterdam, vol. 1, pp. 205-263 (1978). Mll-BORROW. B. V. and R. C. NODDLE: Conversion of 5-{1,2-epoxy 2,6,6-trimethyl(cyclohexyl)3-methyl penta cis-2-trans-4-dienoic acid into abscisic acid in plants. Biochem. J. 119, 727 -734 (1970)_
MILBORROW, B. V. and D. R. ROBINSON: Factors affecting the biosynthesis of ahscisic acid. ]. Exp_ Bot_ 24, 537 -548 (1973)_
NODDLE, R. C. and D. R. ROBINSON: Biosynthesis of abscisic acid: Incorporation of radioactivity from [2_1 4C] mevalonic acid by intact fruit. Biochem.]. 112, 547 - 548 (1969). NORMAN, S. M., R. D. BENNETI, V. P. MAIER, and S. M. POLING: Cytokinins inhibit abscisic acid biosynthesis in Cercospora rosicola. Plant Sci. lett. 28, 255-263 (1983 a). NORMAN, S. M., S. M. POUNG, V. P. MAIER, and E. D. ORME: Inhibition of abscisic acid biosynthesis in Cercospora rosicola by inhibitors of gibberellin biosynthesis and plant growth retardants_ Plant Physiol. 71, 15 -18 (1983 b).
NORMAN, S. M., R. D. BENNETI, S. M. POUNG, V. P. MAIER, and M. D. NELSON: Paclobutrazol inhibits abscisic acid biosynthesis in Cercospora rosicola. Plant Physiol. 80,122-125 (1986). R.~.ll.TON, I. D.: A study of the role of 2,/3-hydroxylation in the control of C-19 gibberellin levels during seedling growth. Z. flir Pflanzenphysiol. 96, 103 -114 (1980). RAIL TON, I. D. and A. K. COWAN: Water stress and the catabolism of (± }-abscisic acid by excised leaves of Hordeum vuLgare. Plant Growth Regul. in press (1987). STEWART, C. R., G. VOETBERG, and P. J. RAYAPATI: Thc cffccts of benzyl adenine, cycloheximide and cordycepin on wilting-induced abscisic acid and salt-induced proline accumulation in barley leaves_ Plant PhysioL 82, 703-707 (1986).
ZrrvAART,]. A. D. and B. V. MILBORROW: Metabolism of abscisic acid and the occurrence of epidihydrophaseic acid in Phaseolus vulgaris. Phytochemistry. 15, 493 - 500 (1976).
1-
Plant Physiol. Vol. 130. pp_273-277 (1987)
Summary The cytokinins, benzyl adenine, kinetin, isopentenyl adenine, and zeatin and the cytokinin
analogue, ancymidol all inhibited the biosynthesis of ahscisic acid from mevalonic acid in mesocarp tissue from ripe fruits of avocado {Persea gratissima}.
Key words: Abscisic acid biosynthesis. cytokinim, ancymidol, inhibition, avocado mesocarp.
Introduction Studies on the production of abscisic acid (ABA) by the fungus Cercospora rosicola, have shown that several cytokinins are potent inhibitors of the biosynthetic pathway to this important sesquiterpenoid plant growth hormone (Norman et aI., 1983 a). Investigations with [l-"C}farnesyl pyrophosphate ([j-1'}FPP) showed thaI one cytokinin, benzyl adenine, (BA), inhibited specifically, post-FPP steps in ABA biosynthesis in this fungus (Norman et a1., 1983 a). In other studies, it was demonstrated that several plant growth retardants and inhibitors of gibberellin (GA) biosynthesis, including ancymidol, which bears structural similarities to cytokinins, would also inhibit ABA production in C. rosicola (Norman et a1., 1983 b). The effect of these compounds on ABA biosynthesis in higher plant tissues has never been investigated. We therefore examined several cytokinins and the cytokinin analogue, ancymidol, as potential inhibitors of ABA biosynthesis in the mesocarp of fruits of Persea gratissima, the only well-documented higher plant system which will synthesize routinely, ABA from radioactive terpenoid precursors such as mevalonic acid (MV A) (Noddle and Robinson, 1969; Milborrow and Robinson, 1973; Cowan and RailIon, 1986). Materials and Methods Plant material Soft, ripe fruits of avocado (Persea gratissima) were purchased from a local trader and used immediately.
Application of compouTJdJ
Fruits were cut in half, de-stoned and the skin removed. Excised blocks of mesocarp (each ZOg) were pre-treated with either ancymidoI (SOO~) or the cytokinins BA, kinetin(K), zeaj. Piant Physinl. Vol. 130. pp. 213-217 (1987)
274
A. K.
COWAN
and I. D.
RAILTON
tin(Z) and isopentenyl adenine(IPA) or adenine (all 500 JLM), each dissolved in Tween 20: acetone :HzO (1: 1: 8, v/v), by infiltration via a series of cuts in the surface of the tissue. A total volume of 0.6 rul of each solution was administered in this way and the tissue blocks were left for 6 hours in continuous illumination (5500 lux) at 25°C in a humid environment under a large inverted crystallizing dish containing wet paper towels.
Biosynthesis 0/ABA This was carried out as described by Milborrow and Robinson (1973). R-{2- '4C}Mevalonolactone (R{2)4C}MVAL), (9.0kBq; sp. act. 1.9 GBq/mmol), purchased from Amersham International, UK, and disssolved in Tween 20: acetone: HzO (1: 1; 8, v/v) (0.2 ml), was administered to each block of avocado mesocarp which had been pre-treated with either ancymidol or each of the cytokinins or adenine for 6 hours. The blocks of mesocarp were then left under the same conditions for a further 24 hours prior to extraction. Extraction and purification ofABA
Tissues were homogenized in ice-cold ethyl acetate: methanol (50: SO, v/v). containing the antioxidant, butylated hydroxy toluene (BHT) (20 rugll), using an Ultraturrax top-drive blender. Each extract was left at 2 °C for 12 hours and was then filtered through Whatman No1 filter paper in a Buchner funnel. The residue was further extracted (2 x) and the combined fil· trates reduced in vacuo at 35°C to a small aqueous volume to which was added 100 m! of 0.5 M KzHP0 4 (pH 8.0). This was partitioned against EtzO (6 x) to remove neutral-basic impurities and then the aqueous phase was re.adjusted to pH 2.5 and further partitioned (6 x) against EtzO to extract the radioactive acids. The EtzO-soluble acid fraction was reduced to dryness in vacuo at 3S oC, dissolved in l.Oml methanol, K,HPO,·IQI,PO, buffer (20mM, pH 8.0) (32,68, v/v), loaded onto a Sep-pak C I8 cartridge (Waters Associates, Milford, MA. USA) and eluted with 6.0 ml of the same solution. The eluate was reduced in vacuo at 35°C and the residue dissolved in a small volume of ethyl acetate: methanol (50: 50, v/ v) for chromatography. Samples were separated by thin-layer chromatography (TIC) on silica gel GF 254 developed (2 x) to 15 em using the solvent system toluene: ethyl acetate; acetic acid (50: 30: 4, v/v) (Zeevaan and Milborrow, 1976). Plates were divided into 30 strips, each strip eluted with methanol and counted in a Beckmann LS5801 scintillation spectrometer using a cocktail of 2,5 diphenyloxazole in toluene (5 gil). Material co-chromatographing with authentic ABA was eluted with HzO-saturated ethyl acetate for further identification.
Characterization ofABA Putative ABA. newly biosynthcsized from R{2- 14C}MVAL was initially identified using the criteria of Milborrow and Robinson (1973) in which label co-migrating with ABA methyl ester (ABAMe) and retained in equal amounts in the diols of ABAMe following reduction of ABAMe with NaBH4. established the radioactive product as ABA. The product. as its methyl ester, was further characterized by radio-gas liquid chromatography (R-GLC). This was carried out on a dual column Perkin-Elmer 990 gas chromatograph interfaced with a Panax Radiogas Detector System as described before (Railton. 1980). Esterified samples were analysed on two separate liquid stationary phases. 2% 5£-30 and 1 % XE-60 on Ga.'ichrom Q (80-100 mesh) which were packed in silanizcd glass columns (1.8 m X 2 mm i.d.) with Ar as carrier gas at a flow rate of 43 ml/min and an oven temperature of 180°C. The effluent carrier gas was split between the flame ionization detector and gas flow proportional counter in a ratio of 1 : 15 and the [ 14C] compound was oxidized to t4C02 over copper oxide at 650°C in a silica furnace tube prior to entry into the proportional counter. The putative, radioactive ABAMe chromatographed as a single peak on both liquid stationary phases and exhib--ited identical retention times to those of authentic ABAMe viz: 2 % SE·30 (Rt - 3.5 min) and 1 % XE-60 (Rt - 3.7 min). Its identity as ABA was further established by u.v. isomerization of its methyl ester followed by analysis on R-GLC as above, when an additional radio.peak with
J. Plant Physiol. VoL 130. pp.
273-277 (1987)
Inhibition of Abscisic Acid Biosynthesis
275
identical retention times to those of t-ABAMe (2% SE-30, Rt == 4.1 min; 1 % XE-60, Rt == 4.7 min) was observed.
Results and Discussion The kinetics of ABA biosynthesis from R{2-"C}MVAL showed that maximum incorporation of label into ABA occurred 24 hours after supplying substrate to mesocarp tissue (Fig. 1). Thereafter, a decline in incorporation into ABA, which was accompanied by the appearance of label in phaseic acid and in the 1',4'-trans diol of ABA (Cowan and Railton, unpublished), probably resulted from ABA catabolism. Thus, in all subsequent feeds, metabolism was allowed to proceed for 24 hours .
•
z
2
"\.
~
o
I>
8z
\.
i
Ii
fig. 1: Kinetics of ABA biosynthesis from R{2_14C]MVAL by excised blocks (20g) of mesocarp from ripe fruits of avocado.
20
40
60
BO
100
TIME (hrs)
Table 1: The effect of cytOkinins and ancymidol on ABA biosynthesis by mesocarp tissue from ripe avocado fruits. Excised mesocarp (20 g) was pre-treated with either BA,K,Z,IPA,adenine or ancymidol (all 500~) for 6 hours prior to the application of R{2- 14C}MVAL (9.0 kBq). Metabolism of substrate was allowed to proceed for 24 hours and biosynthesized ABA was then isolated and characterized as described in «Matcials and Methods,.. All values were corrected for recovery. Treatment
Control Adenine
8A
K
IPA Z
Ancymidol
(Bq)
[ 14 C) Incorporated
(%)
% Inhibition
80.56 78.74 9.83 11.30 13.14 23.42 15.77
0.089 0.087 0.010 0.012 0.014 0.026 0.017
0.00 2.27 87.81 85.97
83.69 70.94 80.43
J. Plant Phy,iol. Vol.
130. pp. 273-277 (1987)
276
A. K. COWAN and I. D. RAILTON
The effect of cytokinins on ABA biosynthesis by avocado mesocarp is shown in Table 1. Whereas the parent molecule, adenine, was essentially inactive, all the N6
substituted purines, which normally exhibit high biological activity in other plant systems (Letham, 1978), markedly inhibited ABA biosynthesis in this tissue. All were supplied at 500 pM which has been reported to inhibit fungal ABA biosynthesis by 80-90% (Norman et al., 1983 a). As in the fungal studies, BA was the most inhibitory to ABA biosynthesis in avocado mesocarp, reducing incorporation of .label by almost 88 %. However, both IPA and K exhibited similar inhibitory activity whereas Z was the least active compound tested. The cytokinin analogue, ancymidol, also supplied at 500 I'M, inhibited ABA biosynthesis markedly in avocado mesocarp tissue
(Table 1). This is the first report that cytokinins and ancymidol can inhibit ABA biosynthesis in higher plant tissues. The steps in ABA biosynthesis in avocado mesocarp which are inhibited by these compounds is currently unknown. Coolbaugh (1984) has shown that both cytokinins and ancymidol can interact with cytochrome P450-mixed function oxidases in GA biosynthesis to inhibit kaurene oxidation in plants. This suggests that these compounds might also affect similarily catalyzed post-FPP oxidations in
fungal and plant ABA biosynthesis. However, studies on fungal ABA biosynthesis have shown that whereas BA does interfere with post-FPP steps (Norman et al., 1983 a), ancymidol appears to inhibit reactions which occur prior to FPP in the terpenoid pathway in this organism (Norman et al., 1986). Likewise, in cell-free extracts of Marah oreganus and shoot tips of Pisum sativum, ancymidol at higher concentrations than those required to inhibit kaurene oxidation, will also inhibit the conversion of
MVA to kaurene which includes several non-oxidative steps common to both ABA and kaurene biosynthesis (Coolbaugh and Hamilton, 1976). Whether or not these compounds exert the same inhibitory effect in all higher plant tissues remains to be investigated. Application of BA to excised barley leaves, for example, did not prevent wilt-induced increases in endogenous ABA levels (Stewart et aI., 1986), a phenomenon believed to involve elevated rates of ABA biosynthesis from MVA (Milborrow and Noddle, 1970). However, recent evidence (Railton and Cowan, 1987) suggests that water stress operates in barley leaves to reduce the catabolism of ABA rather than to increase its biosynthesis which might help explain these apparently anomalous findings. Acknowledgements This work was supported by grants from the CSIR(FRD) Pretoria South Africa to I.D, Railton.
References
R. c.: Inhibition of ent-kaurene oxidation by cytokinins.]. Plant Growth Regul. 3,97 -109 (1984). COOLBAUGH, R. C. and R. HAMILTON: Inhibition of ent-kaurene oxidation and growth by a-cyclopropyl-a[p-methoxyphenyl]-5-pyrimidine methyl alcohol. Plant Physiol. 57, 245-248 (1976). COOLBAUGH,
f Plant PIry,ioi. Vol. 130. pp.
213 - 217 (1987)
Inhibition of Abscisic Acid Biosynthesis
277
COWAN, A. K. and L D. RAILTON: Chloroplasts and the biosynthesis and catabolism of abscisic acid_ J. Plant Growth Regul. 4, 211- 224 (1986)_
lETIiAM, D. S.: Cytokinins. In: LETHAM, D. S., P. B. GOODWIN, T.]. V. HIGGINS (Eds.): Phytohormones and related compounds: Acomprehensive treatise. Elsevier/North Holland Biomedical Press, Amsterdam, vol. 1, pp. 205-263 (1978). Mll-BORROW. B. V. and R. C. NODDLE: Conversion of 5-{1,2-epoxy 2,6,6-trimethyl(cyclohexyl)3-methyl penta cis-2-trans-4-dienoic acid into abscisic acid in plants. Biochem. J. 119, 727 -734 (1970)_
MILBORROW, B. V. and D. R. ROBINSON: Factors affecting the biosynthesis of ahscisic acid. ]. Exp_ Bot_ 24, 537 -548 (1973)_
NODDLE, R. C. and D. R. ROBINSON: Biosynthesis of abscisic acid: Incorporation of radioactivity from [2_1 4C] mevalonic acid by intact fruit. Biochem.]. 112, 547 - 548 (1969). NORMAN, S. M., R. D. BENNETI, V. P. MAIER, and S. M. POLING: Cytokinins inhibit abscisic acid biosynthesis in Cercospora rosicola. Plant Sci. lett. 28, 255-263 (1983 a). NORMAN, S. M., S. M. POUNG, V. P. MAIER, and E. D. ORME: Inhibition of abscisic acid biosynthesis in Cercospora rosicola by inhibitors of gibberellin biosynthesis and plant growth retardants_ Plant Physiol. 71, 15 -18 (1983 b).
NORMAN, S. M., R. D. BENNETI, S. M. POUNG, V. P. MAIER, and M. D. NELSON: Paclobutrazol inhibits abscisic acid biosynthesis in Cercospora rosicola. Plant Physiol. 80,122-125 (1986). R.~.ll.TON, I. D.: A study of the role of 2,/3-hydroxylation in the control of C-19 gibberellin levels during seedling growth. Z. flir Pflanzenphysiol. 96, 103 -114 (1980). RAIL TON, I. D. and A. K. COWAN: Water stress and the catabolism of (± }-abscisic acid by excised leaves of Hordeum vuLgare. Plant Growth Regul. in press (1987). STEWART, C. R., G. VOETBERG, and P. J. RAYAPATI: Thc cffccts of benzyl adenine, cycloheximide and cordycepin on wilting-induced abscisic acid and salt-induced proline accumulation in barley leaves_ Plant PhysioL 82, 703-707 (1986).
ZrrvAART,]. A. D. and B. V. MILBORROW: Metabolism of abscisic acid and the occurrence of epidihydrophaseic acid in Phaseolus vulgaris. Phytochemistry. 15, 493 - 500 (1976).
1-
Plant Physiol. Vol. 130. pp_273-277 (1987)