Plant Physiol. (1987) 85, 916-921 0032-0889/87/85/0916/06/$0 1.00/0

Conversion of Xanthoxin to 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 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 inhibitors and mutants xanthoxin to abscisic acid. The enzyme activity in dialyzed or acetone- which are unable to synthesize 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. Acetone precipitation was done by adding four volumes and a carbonyl, respectively. When the extracts were either of -20°C acetone to 1 vol of undialyzed supernatant. The dialyzed or precipitated with acetone the enzyme activity was solution was centrifuged at 15,000g, the precipitate extracted increased 3- to 4-fold. Heat-inactivated or acid-treated extracts with 50 mm K-phosphate (pH 7.5) and used for enzyme assays. produced no detectable ABA. The enzyme activity in the undi- Enzyme Assay. The assay tubes contained 0.5 to 1.0 jug xan- alyzed extracts showed little dependence on added NADP, but thoxin, 0.5 ml K-phosphate (pH 7.5), 0 to 1 ltmol NADP, and the activity of the dialyzed or acetone-precipitated extracts was 0.25 ml enzyme extract in a total volume of 0.85 ml. There was greatly increased by additions of either NAD or NADP with a control tube for each assay without added xanthoxin, so that endogenous ABA could be subtracted from that formed during Table I. Conversion ofXanthoxin to ABA by Bean LeafExtracts the assay. Incubation was at 28°C for 1 h, and the reaction was stopped by the addition of 90 yd concentrated HCI. 3H-ABA Complete assay contains extract equivalent to 160 mg fresh weight 500 ng 0.25 mm NADP; incubation for 1 h at 28°C. (10,000 dpm. 10 Ci mmol-') (29) was added to the solution tissue, xanthoxin, was to which was chilled and then centrifuged. Water added the Extract Conditions ABA supernatants to bring the HCI to 0.1N, and the solutions were Assay Formed passed through C18 Sep-Pak cartridges (Waters Associates). The ng cartridges were washed with S ml of 20% aqueous methanol and -NADP 47 the ABA was eluted with 5 ml of 70% aqueous methanol con- Undialyzed +NADP 67 taining 0.1 N HCI. This fraction was evaporated to dryness in vacuo and the ABA methylated with ethereal diazomethane (21). Dialyzed Complete 212 -NADP 21 The ABA methyl ester was further purified by HPLC on a 3 um -Extract NDa Spherisorb column (4.6 mm x 15 cm). Elution was isocratic at Heat-killed extract ND 1 ml/min with hexane containing 5% isopropanol. The ABA Acetone Complete 228 and t-ABA methyl esters were separated by HPLC and then precipitated quantitated with a Perkin-Elmer Sigma 2000 gas chromatograph ' Not detected. 918 SINDHU AND WALTON Plant Physiol. Vol. 85, 1987 similar results (Fig. 2). Figure 3 shows that the enzyme activity ofthe xanthoxin to ABA within a 1-h incubation period. Figure is maximal between pH 7.0 and 7.5. Figure 4 shows that there is 5 shows such an instance in which the rate ofconversion appears a linear dependency on the quantity ofleafextract when 500 ng to be linear until more than 80% of the substrate is converted to xanthoxin is incubated for I h with dialyzed extracts equivalent ABA. to between 16 and 160 mg fresh weight of leaf. For most of the The increased activity observed when the extracts were di- results described in this paper, we used tissue extracts correspond- alyzed or precipiated with acetone suggested the presence of an ing to 80 mg leaf fresh weight. When 500 ng xanthoxin was inhibitor(s). After the removal ofacetone, supernatants obtained incubated with 80 mg tissue extract, the production ofABA was from the acetone precipitation were shown to inhibit the pro- usually linear for at least 2 h after a short lag period. Conse- duction ofABA when added to dialyzed extracts. The inhibitory quently, our normal incubation period was 1 h. Occasionally, activity was heat stable and could be partitioned into butanol at extracts were considerably more active and converted almost all either acidic or basic pH. The activity could be further purified 300

;200

cj 0 0 U D 0 a. 0 0.

100

0 1 2 3 200

NADP CONC (M) TISSUE EOUIVALENT ( mg fr Wt ) Fwo. 2. Effects of NADP concentration on the conversion of xan- FIG. 4. Relationship between quantity of dialyzed P. vulgaris leaf thoxin to ABA by dialyzed leaf extracts of P. vulgaris. Conditions as in extract and the conversion ofxanthoxin to ABA. Conditions as in Table Table III. III. ifnu. 500

a a 140v a

a 400 . 120

a

0 c 100 3001 0 0 c

a at 80 < 2001

60

100 .0 40

20 n -. ; 6 7 8 0 100 200 300

pH TIME (min) FIG. 3. Effects of pH on the conversion of xanthoxin to ABA by FIG. 5. Conversion of xanthoxin to ABA by dialyzed. P. vulgaris leaf dialyzed leaf extracts from P. vulgaris. Conditions as in Table III. extracts as a function oftime. Conditions as in Table III. ENZYMIC CONVERSION OF XANTHOXIN TO ABA 919 on an ODS sep-pak. Butenone is a naturally occurring ketone protein(s) must be synthesized prior to the stimulation of ABA with a ring structure identical to that of xanthoxin, but with a synthesis. For this reason, we determined whether the xanthoxin side-chain 2 carbon atoms shorter. Although butenone seemed oxidizing activity was affected either by water stress or by cyclo- like a candidate for an inhibitor, we found that it does not inhibit heximide treatment. Table III shows xanthoxin oxidizing activity the conversion of xanthoxin to ABA, even when present at and ABA levels in stressed and nonstressed leaves of several concentrations considerably higher than we have found it in bean plants. We observed enzyme activity in all of the plants that we leaves (data not shown). Fractionation suggested that much of assayed, although the apparent activity varied considerably. This the inhibitor activity is present in the chloroplasts. The identity widely varying activity may be more apparent than real, however, ofthe inhibitor(s) is currently unknown. since we assayed the enzyme activity using conditions optimized Xanthoxin and t-Xanthoxin as Substrates. Abscisic acid in for the bean leaves. In other experiments we observed no appar- either stressed or turgid leaves occurs almost exclusively as ABA, ent activity in tomato leaves under these conditions, but consid- rather than as its t-ABA isomer. Xanthoxin appears to occur in erable activity was found when dithiothreitol was added to the bean leaves primarily as the trans-isomer, even when care is homogenization medium. Table III shows that there are differ- taken not to isomerize it during isolation (8). Consequently, we ences between the enzyme activities measured in stressed and were interested in comparing the oxidizingactivity ofleafextracts turgid leaves, but that these differences are relatively small and with xanthoxin and t-xanthoxin as substrates. Table II shows the direction of the changes inconsistent. It seems unlikely that that t-xanthoxin is converted to t-ABA by the extracts at a rate the large increases in ABA levels measured in the stressed leaves which is about 40% of that at which xanthoxin is converted to are due to increased xanthoxin oxidizing activities. When bean ABA under the same conditions. ABA is also formed from t- leaves were exposed to cycloheximide at concentrations sufficient xanthoxin but in smaller quantities. We believe this is because to eliminate the subsequent stress-induced increases in ABA the t-xanthoxin always contained 5 to 10% xanthoxin which we levels, there was no apparent effect on the xanthoxin oxidizing were unable to eliminate. When the two isomers were present activity (data not shown). together in the assay at equal concentrations, ABA was formed Xanthoxin Oxidizing Activity in Bean Roots. There are several at a rate almost 3 times that of t-ABA. The conversion of t- reports indicating that ABA is synthesized in roots and accu- xanthoxin to t-ABA was also observed when 2-'4C-xanthoxin mulates when the roots are water-stressed, although to a lesser was fed to tomato plants, although at a reduced rate in compar- extent than occurs in leaves (4, 27). Table IV shows that bean ison with the xanthoxin to ABA conversion (25). roots have xanthoxin oxidizing activity, although the activity is Effects of Water Stress on Xanthoxin Oxidizing Activity in considerably less than measured in the leaves. As is the case with Various Plants. ABA accumulates in the leaves of a variety of the bean leaves, the activity is enhanced several-fold when the mesophytic plants when they are subjected to water stress. Several extracts are precipitated with acetone, suggesting the presence of investigators have reported that cycloheximide, an inhibitor of inhibitor(s). eukaryotic cytosolic protein synthesis, prevents the rise in ABA Subcellular-Localization of Enzyme Activity. The initial report accumulation (5, 19). We have also found that both cyclohex- concerning the localization of ABA biosynthesis indicated that imide and puromycin inhibit ABA accumulation in bean leaves it occurred in the chloroplast ( 14). Two subsequent reports have (data not shown). One explanation for these results is that a suggested, however, that MVA is not converted to ABA by the chloroplasts ofseveral plants (5, 12). Because ofthese conflicting Table II. Conversion ofXanthoxin and t-Xanthoxin to ABA and t-ABA reports we investigated the xanthoxin oxidizing activity in the chloroplasts ofbean leaves. We observed no detectable xanthoxin Assay contains dialyzed extract equivalent to 80 fresh bean mg weight oxidizing activity in the crude chloroplast fraction, even though leaf, xanthoxin, or t-xanthoxin as 1.2 mM indicated, NADP; incubation it contained 74% ofthe activity ofthe chloroplast marker enzyme I h at 28C. NADP-TPDH. Essentially all ofthe xanthoxin oxidizing activity Product Formed was found in the supernatant after sedimenting the crude chlo- Substrate roplast fraction. When a crude extract was subjected to differ- ABA t-ABA ential centrifugation, 90% of the xanthoxin oxidizing activity ng was found in the 100,000g supernatant, appearing in the same 500 ng xanthoxin 151 fraction as the cytosolic marker NAD-dependent TPDH (Table 500 ng t-xanthoxin 13 43 V). 250 ng xanthoxin + 500 ng t-xanthoxin 84 55 DISCUSSION 500 ng xanthoxin + 500 ng Our results t-xanthoxin 120 44 indicate that cell-free extracts from the leaves of several plants are capable of converting xanthoxin to ABA by Table III. Enzyme Activity and ABA Levels in Stressed and Turgid Leaves of Various Plants Acetone-precipated leaf extracts equivalent to 80 mg tissue fresh weight used for enzyme. Assays contain 500 ng xanthoxin, 1.2 mm NADP; incubation I h at 28°C. Leaves stressed for 4 h. Plant Enzyme Activity ABA Levels Rate ofABA Turgid Stressed Turgid Stressed Accumulationa ng ABA/h.g-' fresh ngABA/gfresh wt ng ABA/h-g-' wt fresh wt P. vulgaris 2124 2904 82 2411 666 V. radiata 1428 1285 86 355 77 Z. mays 216 324 45 258 62 C. maxima 1428 1236 30 249 63 P. sativum 144 264 55 884 152 a During the 4 h of H20 stress, assuming a 0.5 h lag before accumulation and no metabolism. 920 SINDHU AND WALTON Plant Physiol. Vol. 85, 1987 Table IV. Xanthoxin Oxidizing Activity in Roots and Leaves ofBean phyll cleavage enzyme whose synthesis or activity is greatly Assay contains extract equivalent to 80 mg fresh weight, 1.2 mm increased in stressed leaves. The resultant xanthoxin produced NADP, 500 ng xanthoxin; 1 h incubation at 28°C. by such an enzyme would be rapidly converted to ABA by the Tissue constitutive xanthoxin oxidizing enzymes. Although xanthoxin ABA Produced can be produced from violaxanthin by lipoxygenase, the effi- ug/gfresh wt gg/mg protein ciency of conversion is low and both t-xanthoxin and butenone Leaf are produced in larger quantities than xanthoxin (10). There Acetone ppt. 7.40 2.05 does not appear to be a xanthoxin isomerase, and it seems likely Root that a specific cleavage enzyme would have to produce xanthoxin Crude extract 0.10 0.19 rather than t-xanthoxin, since the latter is converted to t-ABA. Acetone ppt. 0.24 0.44 Our results also indicate that the xanthoxin oxidizing enzymes are not in an organelle, such as the chloroplast, but are cytosolic. Table V. Distribution ofXanthoxin-Oxidizing Activity, Chi, and If xanthoxin is an ABA precursor, these results indicate that the Marker Enzymes in Subcellular Fractions ofBean Leaves final steps in the pathway are extrachloroplastic. The origin of the xanthoxin, however, could be either chloroplastic or extra- Xanthoxin chloroplastic. Fraction Oxidizing Chi Cytxc Catalase NAD-TPDH Since the conversion of xanthoxin to ABA requires two oxi- Activity dations in different parts of the molecule and the opening of an Crude extract 100 100 100 100 100 epoxide ring, it seems likely that more than one enzyme is 20,00g pellet 8 95 82 80 10 involved and that there are several intermediates formed. We I00,OOOg pellet 1 4 9 20 8 have not observed the accumulation ofany intermediates, which 100,000gsuper 90 1 12 13 95 suggests that they are rapidly converted to ABA. Xanthoxin acid and its esters were the only radioactive compounds isolated other than ABA and its metabolites when '4C-xanthoxin was fed to NADP/NAD-dependent reactions. Although xanthoxin is ap- bean and tomato plants (25). Since xanthoxin acid accumulated parently a naturally occurring compound, and is converted to to a greater extent when t-xanthoxin was fed, and most of it ABA when fed to several plants, its role as a normal ABA appeared to be esterified, it is possible that xanthoxin acid is not precursor has not been established. One of the difficulties in a normal ABA intermediate but a side-product. Milborrow and establishing such a role is that xanthoxin occurs in low concen- Garmston (16) reported that '4C-xanthoxin acid is converted to trations in leaves, and its concentration does not appear to be ABA when fed to tomato plants, but the rate of conversion was affected by the imposition of a water stress which may increase very low. the rate ofABA biosynthesis by more than a hundred-fold. Using Although we have shown that xanthoxin oxidizing activity is procedures which minimize the artefactual production of xan- present in the leaves of several plants, as well as in bean roots, thoxin from xanthophylls during isolation, xanthoxin levels in our results do not enable us to conclude that such activity is part bean leaves were estimated to be 1 to 2 ng g-' fresh weight in of the normal conversion of xanthoxin to ABA. It is possible both stressed and nonstressed bean leaves (8). The rate of ABA that the oxidation of xanthoxin to ABA by the cell-free extracts, synthesis in stressed bean leaves can be as high as 600 ng h-'g-' or that which occurs when xanthoxin is fed to plants, results fresh weight (Table III). The rate of synthesis in turgid leaves from a fortuitous combination of activities whose normal func- may be no more than 1 to 2 ng h-lg-' -fresh weight needed to tions are the oxidation of other compounds. maintain an ABA level of 40 ng g-' fresh weight. As shown in Figure 5 and Table IV, we have measured rates of ABA produc- tion from xanthoxin by the bean leaf extracts of over 7,000 ng LITERATURE CITED h-'g-1 fresh weight, although the rate we usually observed was 1. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase closer to 3,000 ng h-1g-' fresh weight. This rate of production in Beta vulgaris. Plant Physiol 24: 1-15 was obtained when 500 ng xanthoxin was converted completely 2. BRADFORD MM 1976 A rapid and sensitive method for the quantitation of to ABA. As Figure 5 shows, the rate of conversion appeared to microgram quantities of proteins utilizing the principle of protein-dye bind- ing. Anal Biochem 72: 248-254 be constant over almost the entire course of the reaction and 3. BURDEN RS, HF TAYLOR 1970 The structure and chemical transformations of thus independent of the xanthoxin concentration. We do not xanthoxin. Tetrahedron Lett 4071-4074 know, however, what the rates of conversion would be at xan- 4. CORNISH K, JAD ZEEVAART 1985 Abscisic acid accumulation by roots of thoxin levels similar to those observed in vivo. There was no Xanthium strumarium L. and Lycopersicon esculentum Mill. in relation to water stress. Plant Physiol 79: 653-658 apparent inhibition of xanthoxin oxidation at high ABA levels. 5. COWAN AK, ID RAILTON 1986 Chloroplasts and the biosynthesis and catabo- These results suggest the possibility that enzymes are present in lism of abscisic acid. J Plant Growth Regul 4: 211-224 bean leaves with sufficient activity and affinity for xanthoxin 6. CREELMAN RA, JAD ZEEVAART 1984 Incorporation of oxygen into abscisic and its oxidation products to account for the necessary rate of acid and phaseic acid from molecular oxygen. Plant Physiol 75: 166-169 ABA 7. DAVIES BH 1965 Analysis of carotenoid pigments. In TW Goodwin, ed, biosynthesis in stressed leaves if the xanthoxin supply can Chemistry and Biochemistry of Plant Pigments. Academic Press, New York, be maintained. The maintenance of the low xanthoxin levels in pp 489-532 stressed leaves would be due to its rapid turnover. We have found 8. DEVIT MJ 1986 Studies with xanthoxin, a possible precursor to abscisic acid, that bean leaves convert exogenous xanthoxin to ABA so rapidly in Phaseolus vulgaris, L. M.S. thesis. SUNY College of Environmental Science and Forestry, Syracuse, NY that our attempts to load leaves with xanthoxin failed. 9. FIRN RD, RS BURDEN, HF TAYLOR 1972 The detection and estimation of the If xanthoxin is an intermediate on the ABA biosynthetic plant growth inhibitor xanthoxin in plants. Planta 103: 263-266 pathway, our results suggest that its oxidation does not limit the 10. FIRN RD, J FRIEND 1972 Enzymatic production of the plant growth inhibitor rate ofABA biosynthesis in turgid leaves. Our results also indicate xanthoxin. Planta 103: 262-266 1 1. GIBBS M 1955 TPN triosephosphate dehydrogenase from plant tissue. Methods that the xanthoxin-oxidizing enzyme(s) cannot be the protein or Enzymol 1: 411-415 proteins whose syntheses may be required prior to the accumu- 12. HARTUNG W, B HEILMANN, H GIMMLER 1981 Do chloroplasts play a role in lation of ABA in water-stressed leaves. Since the xanthoxin abscisic acid synthesis? Plant Sci Lett 22: 235-242 oxidizing activity needed to maintain ABA levels in unstressed 13. LUCK H 1963 In HU Bergmeyer, ed, Methods of Enzymatic Akxlysi%. A>a- demic Press, New York, pp 885-888 leaves appears to be present in great excess, one possible candi- 14. MILBORROW BV 1974 Biosynthesis of abscisic acid by a cell-free system. date for control of ABA biosynthesis is a hypothetical xantho- Phytochemistry 13: 131-136 ENZYMIC CONVERSION OF XANTHOXIN TO ABA 921 15. MILBORROW BV 1983 Pathways to and from abscisic acid. In FT Addicott, ed, duced by photolysis of violaxanthin. Phytochemistry 9: 2217-2223 Abscisic Acid. Praeger, New York, pp 79-112 24. TAYLOR HF, RS BURDEN 1973 Preparation and metabolism of 2-14-cis,trans 16. MILBORROW BV, M GARMSTON 1973 Formation of (-)-1 ',2'-epi-2-cis-xan- xanthoxin. J Exp Bot 24: 873-880 thoxin acid from a precursor of abscisic acid. Phytochemistry 12: 1597-1608 25. TAYLOR HF, RS BURDEN 1974 The biochemistry of xanthoxin and its rela- 17. MOORE R, JD SMITH 1984 Growth, graviresponsiveness and abscisic acid tionship to abscisic acid. In K Schreiber, HR Schutte, G Sembdner, eds, content of Zea mays seedlings treated with fluridone. Planta 162: 342-344 Biochemistry and Chemistry of Plant Growth Regulators. Halle, German 18. MOORE R, JD SMITH 1985 Graviresponsiveness and abscisic acid content of Democratic Republic, pp 187-196 roots of carotenoid-deficient mutants of Zea mays. Planta 164: 126-128 26. TAYLOR HF, TA SMITH 1967 Production of plant growth inhibitors from 19. QUARRIE SA, PG LISTER 1984 Effects of inhibitors of protein synthesis on xanthophylls: a possible source of dormin. Nature 215: 1513-1514 abscisic acid accumulation in wheat. Z Pflanzenphysiol 114: 309-314 27. WALTON DC, MA HARRISON, P COTE 1976 The effects of water stress on 20. ROBINSON DR, G RYBACK 1969 Incorporation of tritium from (4R)-4-3H- abscisic acid levels and metabolism in roots of Phaseolus vulgaris L. and mevalonate into abscisic acid. Biochem J 1113: 895-897 other plants. Planta 131: 141-144 21. SCHENK H, JL GELLERMAN 1960 Esterification offatty acids with diazomethane 28. WALTON DC, Y Li, SJ NEILL, R HORGAN 1985 Biosynthesis of abscisic acid: on a small scale. Anal Chem 32: 1412-1414 a progress report. In DB Randall, DG Blevins, RL Larson, eds, Current 22. SMITH L 1955 Spectrophotometric assay ofcytochrome C oxidase. In D Glick, Topics in Plant Biochemistry and Physiology 1985. University of Missouri, ed, Methods of Biochemical Analysis, Vol 2. Interscience, New York, pp Columbia, pp 11 1-1 17 427-434 29. WALTON DC, R WELLNER, R HORGAN 1977 Synthesis oftritiated abscisic acid 23. TAYLOR HF, RS BURDEN 1970 Identification of plant growth inhibitors pro- of high specific activity. Phytochemistry 16: 1059-1061