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. The pigments were identified initially by min'. ABA, PA, and DPA were methylated with ethereal comparing their retention times and visible spectra with stand- diazomethane (22). The methyl esters were further purified ards. The identities of violaxanthin, neoxanthin, and anther- on a 4.6 x 150 mm 3 ,um Spherisorb silica column. Elution axanthin were confirmed by their spectral shifts after the was isocratic at 1 ml min' with hexane:isopropanol (95:5) addition of acid (5). The identities of 9'-cis-neoxanthin and for ABA and PA methyl esters and hexane:isopropanol 9-cis-violaxanthin were confirmed by their optical rotatory (90:10) for DPA methyl ester. ABA and its two metabolites dispersion and circular dichroism spectra recorded with Cary were quantitated by GC and the identities of the three com- 60 and 14 recording spectrophotometers, respectively (1, 2, pounds confirmed by GC-MS (12). Small quantities of [3H] 18). The unknown xanthophyll concentrations in eluates from ABA, [3H]PA, and [3H]DPA were added to the samples at the the HPLC column were estimated by comparing areas under beginning of extraction to monitor for purification losses and the peaks obtained at 436 nm with the areas obtained with estimates of all concentrations were corrected for losses. VIOLAXANTHIN IS ABA PRECURSOR 553

Searching for Putative ABA Precursors; Xanthoxin, ABA after a 7 h stress. Consequently, in several instances we have Aldehyde, and Apo-Xanthophylls measured only ABA and PA levels after a 7 h stress period. Leaves which had been detached, but not stressed, were also Detached leaves from dark-grown bean plants were water- analyzed. The results indicate that even leaves from 4 d old stressed for 2 and 14 h in the presence of 1802 Apo-xantho- plants are capable ofproducing ABA at levels similar to leaves phylls were analyzed in the same way as described for the from 7 d old plants, even though the former contain markedly xanthophylls. System A was used for initial screening by lower levels of xanthophylls. In addition, the levels of two HPLC, except that detection was at 370 nm (9). To detect xanthophylls, cis- plus trans-violaxanthin and 9'-cis-neoxan- more polar apo-xanthophylls, system A was modified so that thin, were considerably reduced in stressed plants of all ages, elution was linear from 50% to 80% of the acetoni- but not in the detached non-stressed plants. The sum of trile:isopropanol mixture (85.5:4) over 25 min. Xanthoxin reductions in these two xanthophylls was similar on a molar and ABA aldehyde were looked for in the neutral fraction basis to the accumulation of ABA plus PA. In addition to the obtained by partitioning with ether against the aqueous extract xanthophylls shown in Table II, we also measured a- and f- which had been adjusted to 1% NaHCO3. ABA methyl ester carotene levels. As suggested in Table I, they were very low (1.5 nmol g fresh weight-') was added as an internal standard and did not change significantly during stress. We have never to the ethereal fractions. The ether was removed in vacuo and found them to vary significantly as a result of stress and have the residue dissolved in 20% aqueous methanol. This solution not included their levels in subsequent tables. was added to a C18 Sep-Pak column and xanthoxin, ABA aldehyde, and ABA eluted with 85% aqueous methanol. The Fluridone Treatment eluates were analyzed by GC-MS with conditions as described for ABA methyl ester (12), except that an 8 m SPB- 1 fused Since the levels of xanthophylls in the leaves of 4 d old silica capillary column was used. dark-grown plants were very low, stress-induced reductions in their levels were especially apparent. The leaves ofdark grown RESULTS plants weight only about 1 mg at this age, however, and it was difficult to obtain sufficient material that included the Carotenoids and ABA Levels in Dark- versus Light-Grown necessary kinetic Plants replicates and controls for studies. Since xanthophyll levels were already sufficient by 4 d to support Table I compares the levels of xanthophylls and carotenes significant ABA production, we sprayed the plants with flur- and the levels of ABA, PA, and DPA in stressed leaves from idone, an inhibitor of carotenoid biosynthesis, and allowed plants grown either in the light or dark for 7 days. The levels them to grow for an additional 4 or 5 d. The leaves continued of neoxanthin, violaxanthin, lutein, and A-carotene were to expand, but xanthophyll production was reduced, produc- sharply reduced in the leaves of dark-grown plants. The sum ing leaves with low xanthophyll levels but of a size sufficient of ABA, PA, and DPA produced (we assume this to be for our experiments. Table III shows the effects of this treat- equivalent to total ABA synthesis) during a 7 h stress period, ment on the levels of 9'-cis-neoxanthin and violaxanthin (cis however, was at least comparable to that measured in the plus trans isomers). Table III also shows that the fluridone light-grown plants. In fact, we have found consistently that treatment had no significant effect on the ability ofthe stressed ABA synthesis is higher in dark-grown plants than in light- leaves to produce ABA. By contrast, when seeds were imbibed grown plants during stress. We are unable even to speculate in fluridone solutions and the seedlings sprayed twice daily, on the cause of this apparent difference. the xanthophylls in the 7 d seedlings were almost eliminated. With this treatment, production of ABA was also sharply Seedling Age curtailed in stressed leaves. To the extent that ABA and PA were produced, however, there were comparable reductions Table II shows the results of an experiment to determine in the levels of 9'-cis-neoxanthin and violaxanthin. the age of dark-grown plants at which their leaves are capable of responding to water stress with significant ABA production Kinetics of Xanthophyll Reduction and ABA Accumulation and the xanthophyll levels such leaves contain. Xanthophyll, ABA, and PA levels were measured before and after a 7 h Table IV shows the results of a kinetic experiment in which stress. We have observed little or no change in DPA levels levels of various xanthophylls, as well as levels of ABA, PA,

Table I. Concentrations of Carotenoids and Water Stress-Induced ABA, PA, and DPA in Dark-Grown and Light-Grown Bean Leaves Nonstressed leaves from 7 d plants grown under light and dark were used for analysis of carotenoid contents. The detached leaves were water-stressed for 7 h for analysis of water stress-induced ABA, PA, and DPA.

Treatment Nxa 9'-cis-Nx Vx 9-cis-Vx (?)-cis-Vx LtE Ax Lt Zx Ca Carotenoid ABA + PA + PA nmol g fresh wt-1 Dark-grown 5.1 9.4 50.1 2.8 2.0 12.5 13.8 70.3 7.0 4.1 177.1 16.9 Light-grown 3.6 110.4 94.3 0.8 1.6 14.0 1.8 361.5 11.2 195.8 795.0 12.3 8 Nx, neoxanthin; Vx, violaxanthin; LtE, lutein-5,6-epoxide; Ax, antheraxanthin; Lt, lutein; Zx, zeaxanthin; Ca, carotenes. 554 Li AND WALTON Plant Physiol. Vol. 92, 1990

Table II. Effects of Seedling Age on Xanthophyll and Water Stress-induced ABA Concentrations Plants grown in dark without fluridone. Treatment: none, before detachment; detached, detached for 7 h; stressed, detached and water- stressed for 7 h. Results are means of two to three replicates ±SD.

Age Treatment Nxa 9'-cis-Nx VXb LtE + Ax Lt Total ABA + PA d nmol g fresh wt-' 4 None 2.0 ± 0.3 2.7 ± 0.2 15.8 ± 1.0 6.7 ± 0.4 17.3 ± 0.9 44.5 ± 2.6 0.4c Stressed 1.9 ± 0.2 0.7 ± 0.1 4.3 ± 0.2 5.3 ± 0.3 16.5 ± 1.0 28.7 ± 1.7 15.7 ± 1.2 5 None 3.1 ± 0.2 4.9 ± 0.3 32.0 ± 1.5 13.5 ± 0.7 35.9 ± 1.2 89.4 ± 3.8 0.8 ± 0.1 Detached 3.0 ± 0.2 4.9 ± 0.3 31.0 ± 1.0 12.5 ± 0.6 35.0 ± 1.0 86.7 ± 2.9 1.7 ± 0.2 Stressed 3.0 ± 0.1 2.1 ± 0.1 22.5 ± 0.7 10.1 ± 0.3 33.9 ± 1.2 71.8 ± 2.4 16.9 ± 0.8 6 None 4.5 ± 0.3 6.5 ± 0.4 44.2 ± 1.4 20.1 ± 0.9 62.9 ± 2.4 138.2 ± 5.5 1.2 ± 0.1 Detached 4.4 ± 0.2 6.3 ± 0.3 45.1 ± 1.9 20.1 ± 1.1 59.9 ± 3.3 135.8 ± 6.7 1.9 ± 0.1 Stressed 4.4 ± 0.3 4.1 ± 0.4 36.0 ± 1.2 21.4 ± 1.0 60.2 ± 2.5 126.2 ± 5.2 10.5 ± 0.8 7 None 6.2 ± 0.4 8.8 ± 0.5 57.5 ± 3.0 25.6 ± 1.2 76.6 ± 3.1 174.7 ± 8.0 1.3 ± 0.1 Detached 6.0 ± 0.4 8.6 ± 0.6 57.2 ± 2.0 26.1 ± 1.0 75.6 ± 3.5 173.8 ± 6.9 2.7 ± 0.2 Stressed 6.1 ± 0.4 5.0 ± 0.3 48.2 ± 2.1 24.0 ± 0.8 78.2 ± 3.2 161.7 ± 6.5 14.7 ± 0.5 a Abbreviations as in Table I. b Including cis and trans isomers. c SD < ±0.05.

Table IlIl. Effects of Fluridone on Xanthophyll Levels and Water Stress-Induced ABA + PA Concentrations Plants grown in dark for 7 d. Treatment: none, non-water-stressed; stress, detached and water-stressed for 7 h. Only those xanthophylls are shown whose total reduction is greater than 1 nmol except for lutein whose threshold reduction is 5%. Results are means of two replicates ± SD. Changes in Content Sample Treatment 9'-cis-Nxa Vxb ABA + PA Xanthophylls ABA + PA mmol g fresh wt-1 Control None 9.5 ± 0.7 56.2 ± 2.0 2.0 ± 0.3 0.0 0.0 Stress 4.7 ± 0.4 45.2 ± 2.8 15.5 ± 0.8 -15.8 +13.5 Fluridone None 0.7 ± 0.1 3.6 ± 0.0 0.9 ± 0.1 0.0 0.0 Day 0-7 Stress 0.2 1.0 ± 0.2 4.2 ± 0.3 -3.1 +3.3 Fluridone None 5.0 ± 0.3 36.1 ± 1.0 1.5 ± 0.1 0.0 0.0 Day 4-7 Stress 1.5 ± 0.1 25.4 ± 0.8 16.5 ± 1.0 -14.2 +15.0 a Abbreviations as in Table I. b Includes cis and trans isomers.

Table IV. Kinetics of Xanthophyll Reduction and ABA Increase after Water Stress Plants grown in dark for 8 d with fluridone treatment from d 4 to 8. Only those xanthophylls are shown whose total reduction is greater than 1 nanomol except for lutein whose threshold reduction is greater than 5%. Concentrations of ABA, PA, and DPA glucose esters in some samples were also estimated. No significant amounts of the conjugates were detected. Results are differences between 0 time level and are means of five replicates ± SD.

Stress Time 9'-cis-Nxa 9-cis-Vx Vx XanthophyllTotal ABA PA DPA ABA + PA + DPA min decrease nmol g fresh wt-h increase nmol g fresh wt-' 40 -0.3b'c 0.0 0.7 0.4 0.9 ± 0.1 0.0 0.0 0.9 ± 0.1 55 0.9 ± 0.1 -0.4b 4.1 ± 0.1 4.6 ± 0.2 2.2 ± 0.2 0.0 0.0 2.2 ± 0.2 70 2.3 ± 0.1 0.4 ± 0.1 6.3 ± 0.2 9.0 ± 0.3 3.5 ± 0.3 0.5 0.0 4.0 ± 0.3 90 2.6 ± 0.2 0.7 ± 0.1 8.8 ± 0.2 12.1 ± 0.5 6.7 ± 0.5 0.7 0.0 7.4 ± 0.5 150 3.3 ± 0.3 0.8 ± 0.1 12.9 ± 0.2 17.0 ± 0.6 6.8 ± 0.4 5.0 ± 0.3 0.4 12.2 ± 0.7 270 3.6±0.3 0.9±0.1 17.6± 1.1 22.1 ±1.5 5.8±0.2 13.4±0.6 2.0±0.1 21.2±0.9 420 3.6 ± 0.2 0.9 ± 0.1 21.4 ± 1.1 25.9 ± 1.4 4.3 ± 0.6 15.5 ± 1.0 7.9 ± 0.8 27.7 ± 2.4 a Abbreviations as in Table I. b Increase. C SD < ±0.05. and DPA, were determined between 55 and 420 min after the The results show that 9'-cis-neoxanthin levels were reduced imposition of an 18% water loss on dark-grown leaves. A early in the stress period but then remained constant, while portion of each sample was treated with base to hydrolyze violaxanthin levels decreased throughout the entire 420 min any glucose esters of ABA and its two metabolites, but we did stress period. These experiments were done in triplicate and not observe significant accumulations of any of them. confirmed what had been hinted at in previous experiments, VIOLAXANTHIN IS ABA PRECURSOR 555 i.e. the very low 9-cis-violaxanthin levels were also reduced and ABA accumulation which disappears at longer stress in a pattern similar to that of 9'-cis-neoxanthin. These exper- times. The two experiments also illustrate the close correlation iments also indicated that the rate of loss of xanthophylls was between xanthophyll loss and ABA synthesis, regardless of considerably greater than the production of ABA during the the extent of the two processes. initial 2.5 h of stress. Figure 2 compares the rates of total xanthophyll decrease and total ABA synthesis during the 7-h Cycloheximide Treatment stress period. Since the rate of ABA synthesis appeared to be slightly greater than the rate of xanthophyll disappearance When leaves are pretreated with a cytoplasmic protein synthesis inhibitor, such as cycloheximide, stressed leaves do even at the end of the 7-h experiment, we lengthened the not accumulate ABA (20). Table VI shows the effects of stress time in order to determine whether the imbalance would cycloheximide pretreatment on xanthophyll loss and ABA continue. Table V shows the results of two experiments in production in leaves stressed for 6 h. As expected, there was which leaves were stressed for 14 h. There was an apparent little ABA production in the stressed leaves. Xanthophyll 1:1 relationship on a molar basis between the loss of xantho- losses were also greatly reduced, although there was a reduc- phylls and the production of ABA and its metabolites at the tion in violaxanthin levels. Much of this reduction was offset end of the 14-h period. Experiment 1 provides further evi- by increases in antheraxanthin and zeaxanthin levels which dence ofthe early discrepancy between xanthophyll reduction occurred whether or not the leaves had been stressed. It is possible that these changes were due to xanthophyll cycle activity (27). There was also a small, but reproducible, increase 30 in 9-cis-violaxanthin levels.

Effects of a Nitrogen Atmosphere

When leaves are transferred to a N2 atmosphere after being water-stressed, ABA is not produced, presumably because the 20 cleavage of a xanthophyll requires oxygen (4). We hoped to observe the accumulation of the cleavage substrate when stressed plants were transferred to N2, particularly if the 03 substrate is a 9-cis-xanthophyll. Table VII shows that N2 treatment did not result in the major accumulation of a 10 xanthophyll, although there were small increases in anther- axanthin, zeaxanthin, and 9-cis-violaxanthin. Violaxanthin levels decreased whether the leaves had been stressed or not, and whether the leaves had been pretreated with cyclohexi- mide. The reduction was comparable to that occurring in 0 stressed leaves. Although the HPLC eluate was also monitored 0 100 200 300 400 500 at 280 and 370 nm, we observed no apparent increase in the concentrations of any other compounds after N2 treatment. I lul k lniJi Figure 2. Rates of reductions of xanthophylls and accumulation of 0 Incorporation ABA + PA + DPA in water-stressed leaves. Data are recalculated from those shown in Table IV. O, Reduction of xanthophylls; *, When light-grown bean leaves are stressed in the presence accumulation of ABA + PA + DPA. of 1802, one 180 atom is incorporated into the carboxyl group

Table V. Effects of 14 h Stress on Xanthophylls, ABA, PA, and DPA Plants grown in dark for 8 d with fluridone treatment from d 4 to 8. Only those xanthophylls are shown whose total reduction is greater than 1 nmol except for lutein whose threshold reduction is 5%. Concentrations of ABA, PA, and DPA glucose esters in some samples were also estimated. No significant amounts of the conjugates were detected. Results are means of four replicates ± SD.

Treatment 9'-cis-Nxa 9-cis-Vx Vx Ax XanthophyllTotal ABA + PA + DPA decrease nmol g fresh wt-' increase nmol g fresh wt-' Experiment 1 2 h stress 2.6 ± 0.3 0.9 ± 0.1 11.8 ± 0.6 0.0 15.3 ± 1.0 10.1 ± 0.5 14 h stress 2.6 ± 0.2 1.1 ± 0.1 13.4 ± 0.5 0.8b 17.9 ± 0.8 18.9 ± 1.0 Experiment 2 14 hdetach 0.0 0.5 1.6 ± 0.1 0.0 2.1 ± 0.1 1.9 ± 0.1 14 h stress 3.5 ± 0.2 0.8 21. ± 0.8 1.8 ± 0.1 27.1 ± 1.1 26.7 ± 1.4 aAbbreviations as in Table I. b SD < ±0.05. 556 Li AND WALTON Plant Physiol. Vol. 92,1990

Table VI. Effects of Cycloheximide on Xanthophyll Levels and Water Stress-Induced ABA Formation Plants grown in dark for 9 d with fluridone treatment from d 4 to d 9. Leaves sprayed with cycloheximide (50 Ag/ml) 14 h prior to detachment. Only those xanthophylls are shown whose total reduction is greater than 1 nanomole except for lutein whose threshold reduction is 5%. Results are differences between levels prior to and after a 6 h stress and are means of three replicates ± SD. Treatment Vxa 9'-cis-Vx Ax Zx Total Xanthophyll ABA + PA + DPA nmol g fresh wt-1 6 h, nonstress -2.0 + 0.2b +0.3 +0.6 +1.0 ± 0.1 -0.1 +0.2 6 h, stress -4.0 ± 0.3 +0.2 +0.9 ± 0.1 +1.1 ± 0.1 -1.8 +0.8 ± 0.1 a Abbreviations as in Table I. b +, increase; -, decrease.

Table VIl. Effects of Nitrogen Treatment on Changes in Xanthophylls and ABA Plants grown in dark for 9 d with fluridone treatment from d 4 to d 9. Chi where used sprayed on the leaves at 50 Mg/mL 14 h prior to detachment. Detached nonstressed and stressed leaves were placed in a desiccator which was then evacuated and filled with nitrogen several times. The leaves were kept in air or nitrogen for 6 h. Results are means of three replicates ± SD.

Treatment 9'dis-Nxa Vx 9'-cis-Vx Total + + (?)-cis-Vx LtE Ax Zx Xanthophyll ABA PA DPA nmol g fresh wt-' Detach, air +0.2b.c -2.2 +0.2 0.0 0.0 0.0 0.0 -2.0 +1.8 ± 0.1 Stress, air -2.4 ± 0.1 -13.7 ± 0.8 -0.1 0.0 0.0 0.0 0.0 -16.2 +15.1 ± 0.2 Detach, N2 -1.0 ± 0.1 -11.3 ± 0.8 0.0 -0.3 -1.0 +1.2 ± 0.1 0.0 -12.4 0.0 Stress, N2 -1.2 ± 0.1 -12.0 ± 1.1 +0.7 ± 0.1 -0.3 -1.1 ± 0.1 +0.5 +2.2 ± 0.1 -12.6 0.0 Stress, Chi, N2 -1.0 -13.9 ± 0.7 +0.8 -0.2 -0.8 +1.7 ± 0.1 +2.0 ± 0.1 -11.4 0.0 'Abbreviations as in Table I. b +, increase; -, decrease. C SD < ±0.05.

Table Vil. Incorporation of '80 into ABA and Xanthophylls Dark-grown leaves were from 8 d old plants with fluridone treatment from d 4 to 8. Light-grown leaves were from 8 d old plants grown in the greenhouse at ambient conditions. The detached and stressed leaves were incubated with '802 for either 2 or 14 h. ABA was analyzed by GC-MS as ABA methyl ester. Xanthophylls were analyzed by direct probe MS. Water Stress- Time Tissue Compound Induced %180 ABA + PA + DPA h nmol g fresh wt- 2 Dark-grown ABA 8.9 Ring <5 Side chain 84 Light-grown ABA 5.7 Ring <5 Side chain 87 14 Dark-grown Neoxanthin <5 Violaxanthin <5 Antheraxanthin <5 ABA 26.7 Ring <5 Side chain 79 Light-grown ABA 15.8 Ring <5 Side chain 78 of ABA (12). To determine whether the formation of ABA in little incorporation into the ring oxygens, which is consistent leaves from dark-grown plants is similar to that occurring in with a lack of significant violaxanthin synthesis from anther- light-grown leaves, we stressed both detached dark-grown and axanthin or zeaxanthin during the stress period. light-grown leaves in the presence of 1802 and then analyzed the ABA produced by GC-MS. The results in Table VIII show DISCUSSION that ABA produced in the dark-grown leaves, like that syn- thesized in greened bean leaves, had a considerable incorpo- The use of dark-grown leaves to investigate ABA biosyn- ration of 180 into one carboxyl oxygen atom. There was very thesis has several advantages over the use of light-grown VIOLAXANTHIN IS ABA PRECURSOR 557 leaves. Since the levels of xanthophylls, particularly 9'-cis- reported that the all-trans isomer predominated if saponifi- neoxanthin, are much lower in dark-grown leaves, reductions cation was deleted. Recently there have been reports which following water stress are more obvious. The use of dark- suggest that the major isomer in several leaves is the 9'-cis grown leaves may also result in more reproducible analytical compound (8, 1 1). We find that in both dark- and light-grown results, since the presence of Chl in an extract can increase leaves 9'-cis-neoxanthin is the predominant isomer. If either the rate of xanthophyll destruction or isomerization (8). In 9-cis-violaxanthin or 9'-cis-neoxanthin is an ABA precursor, addition, we have observed the nonspecific loss of xantho- the isomerization required for ABA biosynthesis would have phylls in nonstressed detached light-grown bean leaves, while occurred at the C40 stage. There is no evidence that the in dark-grown leaves xanthophyll levels do not decrease in isomerization is enzymatic, or even direct evidence that neox- unstressed detached leaves. anthin is derived from violaxanthin, although the latter con- The results presented in this paper are consistent with the version has been postulated (13). If ABA is derived from the accumulating evidence that ABA is synthesized from xantho- cleavage of a 9-cis isomer, which had been derived from all phylls (28). We have observed a strong correlation between trans-violaxanthin, it seems likely to us that the isomerization reductions in the combined levels of violaxanthin, 9'-cis- is enzymatic. The rapidity and specificity with which the neoxanthin and 9-cis-violaxanthin and the accumulation of isomerization would have to occur during water stress seems ABA and its major metabolites when dark-grown bean leaves to require an enzyme. The absence oflight during stress would are water-stressed. We believe that our data strongly support also rule out a light-catalyzed isomerization. We have reported the presumption that violaxanthin is an ABA precursor in that ABA produced in response to water stress contains 180 water-stressed dark-grown bean leaves. The reduction in vio- in its ring if the epoxide of violaxanthin had been labeled in laxanthin levels accounted for at least 75% of the ABA situ with 180 via the xanthophyll cycle prior to stress (12). produced, regardless ofthe levels of ABA synthesized and the The labeling in ABA was only about 25% of that in violax- levels ofviolaxanthin in the tissue. The only other xanthophyll anthin which suggested dilution by an unlabeled com- present at a level sufficient to supply most of the stress- pound(s). If either 9-cis-violaxanthin or 9'-cis-neoxanthin is induced ABA is lutein, and we obtained no evidence that it an ABA precursor, our results would be explicable since the is metabolized during water stress. The fact that we observed violaxanthin cycle is restricted to all-trans compounds and almost no incorporation of 180 into the ABA ring also indi- neoxanthin is deepoxidated and reepoxidated very poorly cates that lutein was not a precursor. The results reported by (27). The 180 appearing in ABA would be reduced to the Gamble and Mullet (7) from dark-grown barley also suggested extent that the 180 in violaxanthin was diluted by the unla- a possible precursor role for violaxanthin. beled 9-cis-violaxanthin and 9'-cis-neoxanthin pools. Our data also suggest that 9-cis-violaxanthin and 9'-cis- The results obtained with cycloheximide treatments are neoxanthin are ABA precursors, although the evidence is less consistent with a precursor role for the xanthophylls and convincing. The reductions in their levels are considerably suggest that the cleavage enzyme is synthesized as a conse- smaller and occur only during the early stages of water stress quence of stress. Since neither 9-cis-violaxanthin nor 9'-cis- and ABA accumulation. We suggest several possible expla- neoxanthin levels in cycloheximide-treated leaves increased nations for these results. The formation ofthe two cis isomers significantly during stress, it is conceivable, although not may initially lag behind their isomerization or cleavage, so necessary, that enzymes required for their rapid formation that steady state levels are attained only as the cleavage rate are also synthesized as a consequence of stress. slows. Alternatively, the initial cleavage reactions may con- As indicated above, we had hoped to observe an accumu- sume the accessible pools of either one of the cis isomers lation of the xanthophyll which is cleaved by a dioxygenase which is not further utilized as stress continues. during water stress by exposing stressed plants to a nitrogen One of several questions about ABA biosynthesis has to do atmosphere. We did not observe such an accumulation, al- with the stereochemistry around C-2. It was shown relatively though there was a small increase in 9-cis-violaxanthin levels. early in the work on ABA biosynthesis that this double bond The major change observed was a reduction in violaxanthin was formed trans, requiring an isomerization step at a later levels. We had previously observed that green leaves main- stage of synthesis (21). There have been no reports indicating tained under a nitrogen atmosphere for 2 h showed a consid- that possible C,5 precursors, such as trans-ABA, trans-xan- erably reduced ability to make ABA when subsequently trans- thoxin, or trans-ABA aldehyde, can be isomerized in vivo. ferred to air. We found the same to be true of the dark-grown The latter two compounds produce trans-ABA when fed to leaves although to a lesser extent (our unpublished data). One plants and trans-ABA is either unchanged or metabolized to interpretation of these results is that in the absence of 02, compounds retaining the 2-trans double bond. These results violaxanthin is irreversibly converted to compounds unavail- suggest that a 9-cis-xanthophyll, obtained from an all-trans able for ABA synthesis in the subsequent presence of 02. precursor, might be cleaved to form a Ci5 compound with the Since cleavage presumably cannot occur, it seems likely that necessary 2-cis-ABA configuration. Most of the leaf xantho- a derivative of violaxanthin is formed. We were unable to phylls occur primarily in the all-trans configuration, although observe the formation of such a compound, however, even cis isomers, including the 9-cis have been frequently described though we monitored the HPLC eluate at 280 and 370 nm in (6). There has been some confusion about the stereochemistry addition to the usual 436 nm. of neoxanthin. Cholnoky et al. (2) reported that the initial During the first 2 to 5 h of water stress, the total reduction isolation of neoxanthin had probably been that of 9'-cis- in xanthophyll levels appears to have exceeded total ABA neoxanthin, due to treatments used to saponify Chl. They synthesis by as much as 5 nmol g fresh weight-' (Tables IV 558 LI AND WALTON Plant Physiol. Vol. 92, 1990

a xanthin 0 7

Violaxanthin 9.cli

Xanthoxlc

ABA ABr% so3A aIdehyde Xenthoxin Figure 3. Proposed pathways from violaxanthin to ABA in water-stressed bean leaves. and V). Even if the cis-xanthophylls are excluded, the reduc- studies, Milborrow et al. (14) have concluded, however, that tion in violaxanthin levels exceeds ABA accumulation during the CIO compound is not formed as a consequence ofreactions the first 2 h of stress. By 7 h the reduction of xanthophylls leading to ABA formation during water stress. If 9'-cis-neox- and the synthesis of ABA appear to be comparable, as is the anthin is cleaved to form a C15 ABA precursor, then only one case after 14 h. These results suggest that the rate of xantho- ABA molecule can be formed per xanthophyll molecule. If 9- phyll cleavage exceeds the ability of the rest of the pathway cis-violaxanthin is cleaved, it also seems likely that only one to produce ABA during the early stages of stress. The conse- ABA molecule will be formed, although two are theoretically quence of such an imbalance will be either to increase the possible. Our data show a 1:1 relationship between the total concentration ofnormal intermediates or to accumulate com- synthesis of ABA and the reduction in the levels of violaxan- pounds, either reversibly or irreversibly, which are not on the thin, 9-cis-violaxanthin and 9'-cis-neoxanthin. If the reduc- normal pathway. Since the amount of ABA ultimately pro- tion in these xanthophylls resulted exclusively in ABA syn- duced is equal to the amount ofxanthophylls which disappear, thesis, then only one ABA molecule was formed from each we assume that either a normal intermediate or a compound xanthophyll molecule cleaved. Our results do not enable us which can be reconverted to a normal intermediate has ac- to conclude whether a C10 fragment also resulted from cumulated during the early stages of stress. Two possible cleavage. normal C15 ABA precursors are xanthoxin and ABA aldehyde Figure 3 shows several possible pathways for the conversion (23, 24). It is also possible that the initial cleavage of a of violaxanthin to ABA based on the results presented in this xanthophyll produces an apo-xanthophyll with more than 15 and previous papers (12, 23, 24). Although dark-grown plants carbon atoms. We attempted to determined whether these were used to obtain the data presented in this paper, we compounds had accumulated after a 2-h stress, using the suggest that the results are not restricted to dark-grown plants. techniques described in "Materials and Methods." Our pre- We believe that the similar effects of cycloheximide and liminary results did not indicate significant accumulation of fluridone on ABA biosynthesis in dark- and light-grown leaves xanthoxin and we were not able to observe ABA aldehyde. and the almost identical incorporation of '80 into ABA We were also unable to obtain any evidence for the accumu- support our contention. In addition, we have found that cell- lation of an apo-xanthophyll with more than 15 carbons. A free extracts prepared from dark-grown leaves show an ability search for other possible cleavage products is underway. to convert xanthoxin and ABA aldehyde to ABA similar to Taylor (25) has suggested that two ABA molecules are that of light-grown leaves (our unpublished results). formed from one violaxanthin molecule by dioxygenase ac- We believe that our data demonstrate that violaxanthin is tivity with the consequent formation ofa ClO dialdehyde from an ABA precursor in water-stressed bean leaves. Definitive the central portion of violaxanthin. He reported the accu- evidence for how violaxanthin is converted to ABA is lacking, mulation of compounds related to such a C,O dialdehyde but the proposal shown in Figure 3 can serve as a testable when tomato leaves were stressed. Based on D20 labeling working model. To confirm our proposal, it will be necessary VIOLAXANTHIN IS ABA PRECURSOR 559 to demonstrate the presence of a stress-regulated cleavage following the induction of chlorosis by norflurazon. Z Pflan- enzyme(s) and to determine substrate specificities. This should zenphysiol 14: 35-43 11. Khachik F, Beecher GR, Whittaker NF (1986) Separation, iden- allow us to determine whether 9-cis-violaxanthin and/or 9'- tification, and quantification of the major carotenoid and cis-neoxanthin are cleavage substrates and whether a C15 chlorophyll constituents in extracts of several green vegetables compound is the cleavage product. If the initial product is by liquid chromatography. J Agric Food Chem 34: 603-616 larger than C15, a second cleavage will be required. 12. Li Y. Walton DC (1987) Xanthophylls and abscisic acid biosyn- thesis in water-stressed bean leaves. Plant Physiol 85: 910-915 It will also be necessary to obtain information about the 13. Milborrow BV (1982) Stereochemical aspects of carotenoid bio- interconversion of violaxanthin and neoxanthin, particularly synthesis. In G Britton, TW Goodwin, eds, Carotenoid Chem- with regard to their trans and cis isomers, and how the istry and Biochemistry (Proceedings of the 6th International interconversions are regulated by stress. As indicated in Figure Symposium on Carotenoids, July 1981). Pergamon Press, Oxford, pp 279-295 3, we cannot now conclude how 9'-cis-neoxanthin is formed. 14. Milborrow BV, Nonhebel HM, Willows R (1988) 2,7-Dimethyl- The identification of the compound(s) which apparently ac- octa-2,4-dienoic acid is not a by-product of abscisic acid bio- cumulate during the initial 2 to 3 h of stress should be helpful synthesis. Plant Sci 56: 49-53 in either confirming or modifying parts of the model, as will 15. Moore R, Smith JD (1984) Growth, graviresponsiveness and abscisic acid content of Zea mays seedlings treated with fluri- results obtained with other plants and plant tissues. done. Planta 162: 342-344 16. Moore R, Smith JD (1984) Graviresponsiveness and abscisic acid ACKNOWLEDGMENT content of roots of carotenoid-deficient mutants of Zea mays. Planta 164: 126-128 The authors thank Dr. Gregory Boyer for stimulating discussions 17. Neill SJ, Horgan R, Parry AD (1986) The carotenoid and abscisic and helpful suggestions. acid content of viviparous kernels and seedlings of Zea mays. L. Planta 169: 87-96 18. 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