Plant Physiol. (1990) 92, 551-559 Received for publication June 22, 1989 0032-0889/90/92/0551 /09/$01 .00/0 and in revised form September 25, 1989 Violaxanthin Is an Abscisic Acid Precursor in Water-Stressed Dark-Grown Bean Leaves' Yi Li2 and Daniel C. Walton* Department of Biology, SUNY College of Environmental Science and Forestry, Syracuse, New York 13210 ABSTRACT one atom of 180 into the carboxyl group of ABA. One The leaves of dark-grown bean (Phaseolus vulgaris L.) seed- explanation for these latter results is that a preformed xantho- lings accumulate considerably lower quantities of xanthophylls phyll was cleaved by a dioxygenase to form an aldehyde that and carotenes than do leaves of light-grown seedlings, but they was converted to ABA by dehydrogenases. Li and Walton synthesize at least comparable amounts of abscisic acid (ABA) (12) suggested that at least a portion ofABA was derived from and its metabolites when water stressed. We observed a 1:1 violaxanthin in water-stressed bean leaves based on studies in relationship on a molar basis between the reduction in levels of which the violaxanthin epoxide oxygens were labeled in situ violaxanthin, 9'-cis-neoxanthin, and 9-cis-violaxanthin and the with 180 via the xanthophyll cycle. accumulation of ABA, phaseic acid, and dihydrophaseic acid, One ofthe difficulties in demonstrating a precursor role for when leaves from dark-grown plants were stressed for 7 hours. a major leaf xanthophyll is that these compounds are present Early in the stress period, reductions in xanthophylls were greater at high levels in green leaves compared with the levels to than the accumulation of ABA and its metabolites, suggesting the which ABA and its metabolites accumulate even under water accumulation of an intermediate which was subsequently con- verted to ABA. Leaves which were detached, but not stressed, stress. Small differences in xanthophyll levels between leaf did not accumulate ABA nor were their xanthophyll levels re- samples, either real or due to experimental error, can obscure duced. Leaves from plants that had been sprayed with cyclohex- any reductions that might result from ABA synthesis. Resyn- imide did not accumulate ABA when stressed, nor were their thesis of the precursors could also mask any reductions. xanthophyll levels reduced significantly. Incubation of dark-grown Gamble and Mullet (7) stressed dark-grown fluridone-treated stressed leaves in an '802-containing atmosphere resulted in the barley leaves which contained considerably lower xanthophyll synthesis of ABA with levels of 180 in the carboxyl group that levels than were present in untreated green leaves. They were virtually identical to those observed in light-grown leaves. observed that ABA production was still 25% of normal, even The results of these experiments indicate that violaxanthin is an when xanthophyll levels had been reduced by 99%. They also ABA precursor in stressed dark-grown leaves, and they are used reported that and antheraxanthin to suggest several possible pathways from violaxanthin to ABA. violaxanthin, neoxanthin, levels were reduced during the stress period. Their results were confounded, however, by the fact that xanthophyll levels were also reduced, albeit to a lesser extent, when leaves were detached but not stressed. Thus, they were unable to conclude what the stoichiometry was between xanthophyll reduction The details of the ABA biosynthetic pathway in higher and total ABA synthesis. We report on the results we have plants have remained obscure even though MVA3 is known obtained with bean leaves stressed after detachment from to be a precursor (19) and the stereochemistry of ABA for- dark-grown bean seedlings. In this system, only leaves that mation has been shown to be identical to that of the carote- have been stressed show significant changes in xanthophyll noids (21). Most investigators have been unwilling to conclude content, the levels of ABA produced are at least comparable whether ABA is derived directly from farnesyl pyrophosphate to those in light-grown plants, and the total amount of ABA or from the cleavage ofa xanthophyll (28). Recently, however, produced is closely matched by reductions in the levels of evidence has accumulated that the ABA biosynthetic pathway several xanthophylls. These results enable us to draw infer- involves xanthophyll intermediates. Various corn mutants ences about the origin of ABA. have been described that lack the ability to synthesize carot- enoids and that accumulate little or no ABA (16, 17). Inhib- MATERIALS AND METHODS itors of carotenoid biosynthesis, such as norflurazon and fluridone, also inhibit the accumulation of ABA under some Plant Materials conditions (10, 12, 15, 20). Creelman and Zeevaart (3) re- Phaseolus vulgaris L. cv Blue Lake seeds were surface- ported that leaves stressed in the presence of 1802 incorporated sterilized with a 10% Clorox solution for 30 min and then ' Supported by U.S. Department of Agriculture Competitive Re- imbibed in sterile water for 7 h. The seeds were planted in search Grant 86CRCR 12078. flats in a soil:vermiculite mixture (1:1) and grown in complete 2Current address: Biochemistry Department, University of Mis- darkness at about 23°C for 8 or 9 d. Unless otherwise indi- souri, Columbia, MO 6521 1. cated, plants were sprayed with fluridone (13 mg L-') twice 3Abbreviations: MVA, mevalonic acid; PA, phaseic acid; DPA, daily from d 4 to the end ofthe experiment. Water stress was dihydrophaseic acid. initiated by allowing detached primary leaves to lose 18% of 551 552 Li AND WALTON Plant Physiol. Vol. 92, 1990 their fresh weight. The leaves were wrapped in aluminum foil 100 I and incubated for varying periods oftime at ambient temper- 3 atures. All manipulations were carried out under a photo- graphic safe light. At the end of the treatment period, the 80 - leaves were stored in liquid N2 until extracted. All experiments o< 8 >1 were repeated at least once with two to four replicates per :t- (n 60 - U, treatment. C a) a) Chemicals 10 - Fluridone (98%) was obtained from the Eli Lilly Company, fr 2 Indianapolis, IN. 1802 (96.5%) was purchased from MSD, 20 - 1 ~6 7 Montreal, Canada. [3H]ABA (10 Ci mmol-') was synthesized 4 5 9 (26). [3H]PA and [3H]DPA were purified from bean leaves which had been fed [3H]ABA. Cycloheximide was purchased 0 - from Sigma Chemical Co., St. Louis, MO. 0 1 0 20 30 Time (min) 1802, N2, and Cycloheximide Treatments Figure 1. HPLC chromatogram of xanthophylls from 8 d, dark- For the 1802 experiments detached leaves were placed in a grown, fluridone-treated bean leaves. Solvent system A. 1, Neoxan- desiccator that was evacuated 4 times with alternate additions thin; 2, 9'-cis-neoxanthin; 3, violaxanthin; 4, 9-cis-violaxanthin; 5, (?) of N2. After the fourth N2 addition, approximately 20% of cis-violaxanthin; 6, lutein-5,6-epoxide; 7, antheraxanthin; 8, lutein; 9, the N2 was replaced with l802. Four evacuations and additions zeaxanthin. of N2 were also used in the experiments designed to test the effects of an N2 atmosphere on ABA production. Cyclohexi- known concentrations of authentic standards using an inte- mide solutions (50 ,ug mL-') were sprayed on plants 14 h grating unit of the ISCO-Chem Research Main Program, prior to harvesting in the experiments used to test the effect version 2. We found that recoveries of the various xantho- of cycloheximide on ABA production and xanthophyll loss. phylls were at least 95% as estimated from the recoveries of unlabeled purified xanthophylls subjected to the same purifi- cation procedures. Estimated xanthophyll concentrations Carotenoid Analysis were not corrected for losses. Frozen leaf samples were ground with a mortar and pestle Analysis of ABA, PA, and DPA in acetone containing 15 mg L-' (w/v) butylated hydroxyto- luene. The extract was filtered and the filtrate taken to dryness The aqueous acetone homogenate obtained from frozen in vacuo at 30°C. The residue was dissolved in 25 to 50 mL leaves was taken to dryness in vacuo at 30C. The residue was of 80% aqueous acetone, and 100 ,uL aliquots were chromat- taken up in 1% NaHCO3, then partitioned 3 times with equal ographed by HPLC on a 4.6 x 250 mm 5 ,um Spherisorb volumes of ether. For some samples, the aqueous fractions ODS-2 column. Two solvent systems were used to quantitate were adjusted to pH 11 and kept at 65°C for 2 h to hydrolyze the xanthophylls and carotenes. System A consisted ofa linear any ABA, PA, or DPA glucose esters. The aqueous fractions gradient of water and a mixture of acetonitrile and isopropa- containing ABA, PA, and DPA were adjusted to pH 2.5 with nol (85.5:4) from 88% to 100% of the organic mixture over HCI, then added to a C18Sep-Pak column. ABA, PA, and 25 min at 1.2 mL min-'. This system separated the various DPA were eluted with 75% aqueous methanol. The solutions xanthophylls but was not useful for the carotenes (Fig. 1). were taken to dryness in vacuo at 30C and the residues System B was a linear gradient of aqueous acetone from 75% dissolved in 50% aqueous methanol containing 0.05 N acetic to 100% acetone over 20 min at 0.9 mL min-'. This system acid. This solution was chromatographed by HPLC on a 10 was useful for the carotenes, but did not separate lutein-5,6- x 150 mm 10 ,um ODS Spherisorb column. ABA and its two epoxide from antheraxanthin or lutein from zeaxanthin. Elu- metabolites were separated by a linear gradient of from 25% tion of the various compounds was monitored by their ab- aqueous methanol to 100% methanol over 25 min at 2 mL sorbance at 436 nm.