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Conversion of Xanthoxin to Abscisic Acid by Cell-Free Preparations from Bean Leaves' Received for Publication April 14, 1987 and in Revised Form August 21, 1987 RAM K

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Conversion of Xanthoxin to Abscisic Acid by Cell-Free Preparations from Bean Leaves' Received for Publication April 14, 1987 and in Revised Form August 21, 1987 RAM K Plant Physiol. (1987) 85, 916-921 0032-0889/87/85/0916/06/$0 1.00/0 Conversion of Xanthoxin to Abscisic Acid by Cell-Free Preparations from Bean Leaves' Received for publication April 14, 1987 and in revised form August 21, 1987 RAM K. SINDHU AND DANIEL C. WALTON* Department ofBiology, State University ofNew York, College ofEnvironmental Science and Forestry, Syracuse, New York 13210 ABSTRACT produce xanthoxin from violaxanthin at least suggested the possibility of a nonphotolytic origin for xanthoxin (10). Cell-free extracts from the leaves of Phaswolus vulgaris L. convert Recent work (17, 18) with carotenoid inhibitors and mutants xanthoxin to abscisic acid. The enzyme activity in dialyzed or acetone- which are unable to synthesize carotenoids has suggested a ca- precipitated extracts shows a strong dependence on either NAD or rotenoid origin for ABA in plants. Creelman and Zeevaart (6) NADP. The enzyme activity appears to be cytosolic with no significant have obtained evidence that ABA may be derived from the activity observed in chloroplasts. The activity was observed in extracts oxidative cleavage of a xanthophyll already containing the ring from roots of Phawolas vulgaris, and also in extracts prepared from the oxygens found in ABA. Walton et al. (28) have suggested that leaves of PisEm sativim L., Zea mays L, Cuwurbita maxima Duchesne, ABA is derived at least in part from violaxanthin based on 'IO and Vigna radiata L. Neither water stress nor cycloheximide appear to labeling experiments. These results are consistent with, although significantly affect the level ofenzyme activity in leaves. No intermediates clearly not proof of, a role for xanthoxin as an intermediate in between xanthoxin and abscisic acid were detected. ABA synthesis. Despite the evidence that exogenous xanthoxin can be converted to ABA in vivo, we know of no report of its conversion to ABA by cell-free extracts. The work described in this paper was undertaken to obtain information about the conversion of xanthoxin to ABA by cell-free extracts obtained from turgid and water-stressed leaves. The plant hormone ABA is a sesquiterpene, and like other sesquiterpenes, has been shown to be derived from MVA2 (20). MATERIALS AND METHODS The stereochemistry of ABA formation from MVA appears to Plant Material. Seeds of Phaseolus vulgaris L. cv Blue Lake, be identical to that ofthe carotenoids (15). Surprisingly, however, were germinated and grown in a soil-vermiculite mixture (1:1 v/ it is still not possible to make any other definitive statements v) in the greenhouse at ambient conditions. Fully expanded about the biosynthesis of ABA in plants. Not only do we not primary leaves from 2 to 3 week old plants were used for the know the details of the pathway, we cannot even conclude experiments. For water stress experiments, leaves were detached whether ABA is made directly from the usual sesquiterpene and allowed to lose about 12% of their fresh weight. The leaves precursor, farnesyl pyrophosphate, or is derived from the oxida- were then wrapped in aluminum foil and stored at about 22°C tive cleavage of a carotenoid such as violaxanthin (Fig. 1). The for varying periods of time. The entire root systems of 2 week idea that ABA might be derived from carotenoids arose from the old bean plants were used for preparing enzyme extracts. Zea similarity in their structures. Unlike most other known plant mays L. cv Hybrid Yellow, Vigna radiata L. cv Berkin, Cucurbita sesquiterpenes, ABA resembles the cyclic end of carotenoids, maxima Duchesne cv Waltham butternut, and Pisum sativum particularly the oxygenated carotenoids known as xanthophylls. L. cv Wando were grown and stressed in the same manner except The initial experiments exploring the possible link between xan- that leaves were harvested from 3-week old plants. thopltylls4nd ABA involved the irradiation ofpigments obtained Preparation ofXanthoxin. Xanthoxin was obtained by the zinc from nettles (26). This treatment produced a mixture of com- permanganate oxidation of violaxanthin and neoxanthin (3) pounds which had growth inhibitory activity as measured by a which had been isolated from spinach leaves. The initial steps in cress seed bioassay. Purification of the irradiation products the purification of violaxanthin and neoxanthin were done as showed that the inhibitory activity was due to a C- 15 compound described by Davies (7). The saponified xanthophylls were sub- which was given the name xanthoxin. This compound which jected to flash chromatography on a 2 x 20 cm column packed could be obtained by irradiation of violaxanthin, neoxanthin, with 40 ,um ODS (J. T. Baker) after they had been separated and antheraxanthin was subsequently shown to occur naturally from the other carotenoids by partitioning between petroleum in a variety of plants and plant parts (9, 23). ABA levels in ether and 90% aqueous methanol. Viola,anthin and neoxanthin tomato plants were increased by 7-fold when they were fed were eluted from the column with 80% aqueous methanol. The xanthoxin, and '4C-xanthoxin was converted to '4C-ABA and two pigments were dissolved in acetone (1.25 ml acetone/mg) several of its metabolites by tomato and bean plants (24). Since and zinc permanganate (1.5 mg/mg pigment) added over 1 h ABA is produced in plants kept in the dark, it seemed unlikely with vigorous shaking. The solution was then filtered, the acetone that ABA could arise from xanthophyils exclusively by photoox- removed in vacuo, and the aqueous phase partitioned against idation. The discovery that soybean lipoxygenase could also et'her. After removal of the ether, the residue was taken up in 50% aqueous methanol and applied to the flash chromatography 'Supported by National Science Foundation Grant PCM 8219122. column. Xanthoxin was eluted from the column with 50% 2 Abbreviations: MVA, mevalonic acid; TPDH, triose phosphate de- aqueous methanol and then chromatographed on a Whatman hydrogenase. ODS-2 column (10 mm x 25 cm). Elution was with a linear 916 ENZYMIC CONVERSION OF XANTHOXIN TO ABA 917 H3C\OH equipped with a 3H-electron capture detector. The samples were HO CH3_C OH chromatographed isothermally at 195°C on a 6 foot x 0.25 inch Mevalonic acid glass column packed with 3% OV-1 on 80/100 Gas Chrom Q. A portion of the sample was subjected to scintillation counting to determine the recovery of the added 3H-ABA. The identity of the ABA produced enzymically was confirmed by GC-MS. i1 Subcellular Fractions. For the preparation of crude chloro- plasts, bean plants were destarched for 3 d in the dark and then P returned to the light for 3 h immediately before isolation. Fifteen II aPP 04 C02H g of primary leaves were homogenized for 15 s at maximum 02 speed in a Waring Blendor with 100 ml ice-cold 0.5 M sorbitol Farnesyl pyrophosphate F Abscisic acid 015 in 2.5 mm tricine-NaOH (pH 7.6). The brei was filtered through eight layers of cheesecloth and crude chloroplasts were obtained by centrifugation at 10OOg for 10 min. The pellet was resus- pended in 0.05 M K-phosphate (pH 7.5). A portion of this solution was dialyzed against 0.02 M KPO4 (pH 7.5), and then .1\ used to assay xanthoxin oxidizing activity. Carotenoids CHO HO The mitochondrial, peroxisomal, and cytosolic fractions were XanthoxmniCI prepared from bean leaves ground with a mortar and pestle in ,-; 0.33 M sorbitol containing 25 mM tricine-NaOH (pH 7.6). The homogenate was passed through eight layers of cheesecloth and then subjected to differential centrifugation at 20,000g for 15 min and at 100,000g for 60 min. Cyt c oxidase, catalase, and NAD- and NADP-TPDH were 01 to Luck and Gibbs HO2 estimated according Smith (22), (13), (11), Vi\olaxnthin 0C40 respectively. Protein and Chl were estimated by the methods of Bradford (2) and Arnon (1), respectively. FIG. 1. Two possible generalized pathways for the conversion ofMVA GC-MS. GC-MS was done with a Finnegan 4000 GC/MS/DS to ABA. system. For ABA, the GC was done on a SPB-1 fused silica capillary column 30 m x 0.25 mm i.d. with a film thickness of gradient of 50 to 99% aqueous methanol over 15 min at a flow 0.25 ,um (Supelco, Bellefonte, PA). The carrier gas was helium rate of 3.2 ml min-'. This column separated xanthoxin from at 1 ml min-'. After a 2-min hold at 50°C, the oven temperature butenone, a major oxidation product. Xanthoxin was further was increased linearly at 10°C min-'. Xanthoxin was chromat- purified by HPLC on a Spherisorb 3 gm silica column (4.6 mm ographed on an 8 m x 0.32 mm i.d. column at a helium flow x 15 cm). Elution was isocratic at 1 ml min-' with hexane rate of 2 ml min-'. The other conditions were identical to those containing 6.5% isopropanol. t-Xanthoxin and xanthoxin were used for ABA. separated on this column. The yield of xanthoxin was increased by isomerising the t-xanthoxin with light. The identity of the xanthoxin was confirmed by GC-MS. RESULTS Extract Preparation. Tissues were homogenized with a mortar Xanthoxin Oxidizing Activity in Leaves. The data in Table I and pestle in 50 mm K-phosphate (pH 7.5) (3 ml buffer/g tissue). show that undialyzed extracts prepared from bean leaves convert The extract was passed through four layers of cheesecloth and xanthoxin to ABA. This activity will be referred to as xanthoxin then centrifuged at 12,000g for 20 min. The supernatant was oxidizing activity, since the conversion requires that the side- dialyzed against 0.02 M KPO4 (pH 7.5) and used for enzyme chain carbonyl and the ring hydroxyl be oxidized to a carboxyl assays.
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