Optically Pure Abscisic Acid Analogs-Tools for - Plant Physiology

Plant Physiol. (1992) 99, 501-5070032-0889/92/99/0501 /07/$01 .00/0

Received for publication October 29, 1991Accepted January 7, 1992

Optically Pure Abscisic Acid Analogs-Tools for RelatingGermination Inhibition and Gene Expression in

Wheat Embryos1

M. K. Walker-Simmons*, Robert J. Anderberg, Patricia A. Rose, and Suzanne R. AbramsU.S. Department of Agriculture/Agriculture Research Service, Wheat Genetics, Quality, Physiology, and DiseaseResearch, 209 Johnson Hall, Washington State University, Pullman, Washington 99164-6420 (M.K. W. -S., R.J.A.);

and Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon,Saskatchewan S7N OW9, Canada (P.A.R., S.R.A.)

ABSTRACT

We report an examination of the structural requirements of theabscisic acid (ABA) recognition response in wheat dormant seedembryos using optically pure isomers of ABA analogs. Thesecompounds include permutations to the ABA structure with eitheran acetylene or a trans bond at C-4 C-5, and either a single ordouble bond at the C-2' C-3' double bond. (R)-ABA and the threeisomers with the same configuration at C-l' as natural ABA werefound to be effective germination inhibitors. The biologically ac-tive ABA analogs exhibited differential effects on ABA-responsivegene expression. All the ABA analogs that inhibited germinationinduced two ABA-responsive genes, wheat group 3 lea and dhn(rab). However, (R)-ABA and (S)-dihydroABA were less effectivein inducing the ABA-responsive gene Em within the time thatembryonic germination was inhibited.

The plant hormone ABA is a potent germination inhibitorof dormant seed embryos of wheat. To investigate the ABAperception process in embryonic tissue, we have comparedthe effects of naturally occurring (S)-ABA and a series ofoptically pure ABA analogs. The analog series was designedto incorporate systematic permutations to the ABA moleculethat had previously been found to elicit immunological rec-ognition by ABA monoclonal antibodies (28). In this report,we describe the use of these compounds to probe the stereo-chemical requirements of the ABA recognition response forgermination inhibition and for regulation of ABA-responsivegenes.Dormant seed embryos of wheat make a particularly good

system for examining ABA response mechanisms becausethese embryos are so highly responsive to ABA. In wheatseeds, dormancy is not caused by true embryonic dormancy,but is associated with embryonic responsiveness to externalinhibitors such as ABA (16, 26). Low concentrations (0.5-5.0AM) ofABA supplied to embryos isolated from dormant seedblock embryonic germination. As seed dormancy is lost

'Contribution from the U.S. Department of Agriculture, Agricul-tural Research Service, and the College of Agriculture and HomeEconomics Research Center, Department of Crop and Soil Sciences,Washington State University, Scientific Paper No. 9101-51.

during postharvest storage, embryos lose responsiveness toABA, and far higher ABA concentrations (50-500 /LM) arerequired to block germination of embryos from nondormantseeds (25, 26).The application of the low ABA concentrations that block

germination of dormant seed embryos results in the enhance-ment and prolonged expression of a set of ABA-responsivegenes in the growth-arrested tissue (15). These ABA-respon-sive genes include dhn (rab) (4, 17), wheat group 3 lea (6),and Em (1 1). The function of these ABA-responsive geneproducts and their relationship to the maintenance of dor-mancy in seed embryos has not been established. In thisreport, we have employed the array of ABA analogs to deter-mine the shape of the ABA molecule required to inhibitgermination and to induce ABA-responsive genes in dormantseed embryos. Specifically, we sought to probe the stereo-chemical requirements of the ABA molecule at the C-1'position.ABA exhibits optical activity because of the asymmetric

carbon at position 1'. Most physiological studies examiningABA effects have been conducted with a synthetically pro-duced 1:1 racemic mixture of natural ABA, the (S)-(+)-form(structure 1 in Fig. 1), and the nonnatural isomer, (R)-(-)-ABA (structure 2 in Fig. 1). (S)-ABA has been isolated fromplant and fungal sources (30). Both enantiomers have beenobtained in optically pure form by total synthesis (9) and bya number of chromatographic methods for resolving racemicABA and ABA derivatives (30). We have scaled up theresolution of racemic methyl ABA using a chiral analyticalHPLC column (20) to provide sufficient quantities of both(S)- and (R)-ABA for biological testing (7).Only a limited number of studies have reported compari-

sons of the biological activity of (S)- and (R)-ABA. The twoforms have often been found to have equivalent biologicalactivity (29), although the (R)-enantiomer has been found tobe less effective than (S)-ABA in inhibiting root growth ofbarley seedlings and in inducing stomatal closure (5, 24).Other comparisons of (S)- and (R)-ABA have included therates of uptake and metabolism of the two enantiomers. (R )-ABA is found to be more persistent than (S)-ABA in culturefiltrates of bromegrass cell cultures (2). In sunflower embryos,the rates of uptake ofthe (S)- and (R)-ABA enantiomers werefound to be equivalent (3), but differences in uptake have

501

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WALKER-SIMMONS ET AL.

84, 9'

4- *OH CO4@ 2

"'OH OH

4OCH

3,

5

7

OH COOH

9

OH COOH

2

OH COOH

4

8

Figure 1. Structure of (S)ABA and the ABA analogs. Left, Com-pounds with the same absolute stereochemistry as (S)-ABA (1); right,compounds with (R)-ABA (2) stereochemistry.

been observed with bean root segments (14). Metabolism of(S)- occurs more rapidly than with (R)-ABA and yields dif-ferent products (3, 12). There have not been comparisons ofthe effects of (S)- and (R)-ABA on gene expression.How both the (S)- and (R)-enantiomers of ABA can have

effectively equal biological activity is not known. Milborrow(1, 13) has proposed a model of the steric requirements of anABA receptor, which accounts for the observations that bothenantiomers are active in growth inhibition assays. It was

proposed that in the active site, ABA adopts a conformationwith the side chain equatorial so that the C-7' and C-8'methyl groups both have similar equatorial orientations. Thenthe C-7' of (R)-ABA could fit into the space normally occu-

pied by C-8' of natural (S)-ABA.Theories on spatial requirements of the ABA receptor will

be highly speculative until ABA receptors have been isolatedfrom plants and can be studied. However, with our improvedcapability to produce ABA analogs with defined geometryand stereochemistry, we are now in a better position to probethe stereochemical requirements of the ABA perception re-

sponse in plant tissues. Because ABA receptors are not yet

available, we chose in previous work (28) to examine mono-clonal antibodies raised against (S)-ABA as a type of ABAreceptor model system. We determined that a limited numberof optically pure ABA analogs have structures similar enoughto (S)-ABA to elicit recognition by (S)-ABA monoclonalantibodies. Those analogs have simple substitutions to theABA molecule and include ABA analog 3 (Fig. 1) with twohydrogens added to the double bond of the ring so that theC-2' methyl group is cis to the alkyl side chain and ABAanalog 5 (Fig. 1) with a triple bond replacing the C-4 C-5trans double bond of the side chain (27, 28). Only the analogswith the same absolute stereochemistry at C-1' as (S)-ABAare recognized by the monoclonal antibodies.For this report, we first synthesized a set of eight ABA-

related compounds incorporating the two permutations oftheABA molecule that elicited immunological recognition (28).The compounds are shown in Figure 1 in the (S)-ABA series(left) and the (R)-ABA series (right). Second, we have assayedthese compounds for biological activity as germination inhib-itors of dormant seed embryos. Third, we have determinedthe effects of the biologically active ABA analogs on expres-sion ofthe three ABA-responsive genes dhn (rab), wheat group3 lea, and Em. Finally, we have shown that the biologicallyactive ABA analogs do not act by altering endogenous (S)-ABA levels.

MATERIALS AND METHODS

Plant Material and Germination Assay

Dormant seeds of the soft, white, winter wheat (Triticumaestivum L. cv Brevor) were produced as previously described(15). Embryos were dissected from the grain with a razorblade. Small amounts of endosperm and pericarp adhered tothe embryos, which did not prevent the isolated embryos fromgerminating readily in water. Three replicate germinationassays were conducted on 10 dissected embryos each at 30°Cand a weighted germination index calculated as previouslydescribed (26). Solutions of (S)- and (R)-ABA and all methylester ABA derivatives were prepared by dissolving samples ofknown weight (approximately 2 mg) into 200 gL ethanol andthen diluting them into water. All other ABA analogs weredissolved in 400 gL of 1% (w/v) NaHC03, neutralized with100 usL 0.25 M HCl, and diluted into water (1 mL finalvolume).

Synthesis of Optically Pure ABA Analogs

(S)-ABA (structure 1) and the ABA analogs 2 to 8 (Fig. 1)were all obtained from their respective methyl esters la to 8a(methyl esters designated as a are not shown in Fig. 1). (S)-and (R)-methyl abscisate la and 2a were separated fromracemic methyl abscisate by HPLC employing a semi-prepar-ative column. The esters were hydrolyzed to 1 and 2 asreported elsewhere (7).

Syntheses of the 2',3'-dihydroABAs 3 [(+)-(2Z,4E)-(1S,6S)-5-( 1-hydroxy-2,2,6-trimethyl-4-oxocyclohexyl)-3-methyl-2,4-pentadienoic acid] and 4 [(-)-(2Z,4E)-(lR,6R)-5-(1-hy-droxy-2,2,6-trimethyl-4-oxocyclohexyl)-3-methyl-2,4-penta-dienoic acid] and their methyl esters 3a and 4a have beenpreviously reported (10).

502 Plant Physiol. Vol. 99, 1992



OPTICALLY PURE ABA ANALOGS

The methyl esters 5a and 6a and dihydroacetylenic acids 5[(+)-(2Z)- 1 S,6S)-5-(1 -hydroxy-2,2,6-trimethyl-4-oxocy-clohexyl)-3-methylpent-2-en-4-ynoic acid] and 6 [(-)-(2Z)-(1R,6R)-5-(1 -hydroxy-2,2,6-trimethyl-4-oxocyclohexyl)-3-methylpent-2-en-4-ynoic acid] were synthesized by methodsdescribed previously (10). The dihydroacetylenic acid 5 gavethe following: melting point 149 to 151C (chloroform/hex-ane); [aID + 18.90 (c 1.07 CH30H); 'HNMR (360 MHz) CDC136: 6.03 (q, J = 1.4 Hz, 1H, H-2), 2.82 (d, J = 14.4 Hz, 1H,H-5'ax), 2.45 (dd, J= 15.4, 15.3 Hz, 1H, H-3'ax), 2.25 to 2.35(m, 3H, H-3'eq, CHCH3), 2.09 (d, J = 1.4 Hz, C-3 CH3), 2.08(dd, J = 14.4, 2.4 Hz, 1H, H-5',), 1.21 (s, 3H, C-6' CH3),1.14 (d, J = 6.2 Hz, C-2' CH3), and 0.99 (s, 3H, C-6' CH3);IR (CHC13) 3600, 1720 to 1680, 1610 cm-'; GC/electronimpact MS of methyl ester 5a: 278 (3), 261 (2), 219 (47), 207(35), 179 (43), 137 (100); exact mass calculated for C,6H2204278.1518; found 278.1514. The enantiomer 6 and 6a gaveNMR, MS, and IR spectra identical to those for 5 and 5a,and gave melting point 152 to 154°C; exact mass for 6a found278.1520; and [a]D - 20.5 (c 1.18 in CH30H).The syntheses of the optically active 4,5-acetylenic analogs

7, 7a, 8, and 8a has been reported elsewhere, (21). Briefly, achiral derivative of oxoisophorone was alkylated with thelithium salt of methyl (Z)-3-methylpent-2-en-4-ynoate, andthe resultant diastereomeric mixture separated chromato-graphically. The optically pure diastereomers were hydrolyzedto the esters 7a [C]D + 238.3 (c 1.26 in CH30H) and 8a [aCD- 225.0 (c 1.33 in CH30H) and then to the acids 7 [(+)-(2Z)-5-(1-hydroxy-2,6,6-trimethyl-4-oxocyclohex-2-enyl)-3-methyl-2-penten-4-ynoic acid] [aID + 283.5 (c 0.45 inCH30H) and 8 [(-)-(2Z)-5-(1 -hy-droxy-2,6,6-trimethyl-4-oxocyclohex-2-enyl)-3-methyl-2-penten-4-ynoic acid][a]D- 278.8 (c 1.67 in CH30H).ABA analog 9 was synthesized as previously described (10).

Northern Analysis of Transcripts

Embryos were placed on blotter paper in Petri dishes (100x 15 mm) containing 6 mL of sample solution and incubatedin the dark at 20°C as previously described (15). At theindicated times, embryos were dissected and frozen in liquidN2. Poly(A)' RNA was isolated (15) and denatured (22).Northern analyses and fluorography were conducted as pre-viously described (15) with probes prepared from gel-isolatedcDNA inserts (8). Blots were quantified using a radioanalyticimaging system (Ambis, San Diego, CA).

ABA Assay

Dissected embryos were washed three times with 1% (w/v)sucrose, weighed, frozen in liquid N2, and lyophilized. Thelyophilized embryos were weighed and then (S)-ABA wasextracted and assayed by immunoassay (27) using an ABA-BSA-C4' conjugate. The ABA monoclonal antibody 15-I-C5(Idetek, Inc.,2 San Bruno, CA) was used for all ABA measure-ments except for embryos treated with analog 5, where themonoclonal antibody MAC 62 (19, 28) was used.

2 Mention of a specific product name by the U.S. Department ofAgriculture does not constitute an endorsement and does not implya recommendation over other suitable products.

RESULTS

Biological Activity of (S)-ABA and the ABA Analogs asGermination Inhibitors

Optically pure isomers (Fig. 1) of ABA (1 and 2) and theABA analogs dihydroABA (3 and 4), dihydroacetylenic ABA(5 and 6), and acetylenic ABA (7 and 8) were synthesized asdescribed in "Materials and Methods." Biological activity ofnatural ABA (1) and the seven analogs (compounds 2-8) asgermination inhibitors was measured using dormant seedembryos. The two enantiomers of ABA, (S)-ABA (1) and(R)-ABA (2), have equivalent activity as germination inhibi-tors, with about 1 uM causing 50% inhibition of germination(Fig. 2). The resolved isomers of 2',3'-dihydroABA 3 and 4gave very different results. Compound 3, with the same con-figuration at C- 1' as natural ABA, inhibits germination, witha concentration of 5 ,uM causing 50% inhibition. In contrast,dihydroABA 4, analogous to (R)-ABA (2), is virtually inactiveover the entire range of concentrations tested. This sametrend is observed for the other set of 2',3'-dihydroABAs 5and 6. A l-Mm concentration of the 2',3'-dihydroacetylenicacid 5, which has the same absolute configuration at C- ' as(S)-ABA, was just as effective as 1 uM (S)-ABA in inhibitingthe germination index by 50%. The enantiomer 6, like 4, wasinactive.The remaining pair in the set of permutations to the ABA

molecule, acetylenic acids 7 and 8, were inactive as germina-tion inhibitors of embryos (data not shown). These com-pounds and the related racemic mixture are found to beunstable in solution, and can rearrange to form a previouslydescribed lactone (18). The lactone was synthesized and foundto be inactive in germination inhibition (data not shown).

Because the methyl esters of 7 and 8 are chemically morestable, and the methyl abscisate is highly active as a germi-nation inhibitor (29), the germination inhibition of all eightmethyl esters (structures la-8a) of the analogs was assessed tobetter compare the effects resulting from the four changes tothe ABA molecule. The results presented in Table I show thesame trends for the methyl esters (structures la-6a) as shownfor the free acids of those compounds (Fig. 2). The methylesters (structures 7a and 8a) of acetylenic ABA inhibit ger-mination in a manner similar to (S)- and (R)-ABA in thatboth enantiomers are biologically active. In contrast, only theoptical isomer with the (S)-ABA-like configuration is biolog-ically active in the two analog pairs, 3a and 4a, and Sa and6a, with the saturated six-membered ring.To examine further the role of the methyl groups in satu-

rated six-membered rings, we synthesized the (S)-ABA analog9 with the C-2'methyl trans to the ring. This compound,which resembles dihydroABA 3 except that the ring methylis trans to the side chain, did not cross-react with monoclonalantibodies and proved to be ineffective as a germinationinhibitor (data not shown). Also, we synthesized analogs witheither no ring methyls or two methyl groups at the C-2' andC-6' positions. None of these symmetrical analogs had anysignificant biological activity as germination inhibitors (datanot shown). The methyl groups on the ring are necessary forrecognition, and the stereochemical requirements at the C-2'carbon are quite rigid.

503




Table I. Comparison of the Free Acids and Methyl Esters of(S)-ABA and the ABA Analogs as Germination Inhibitors

Compound Free Acid Methyl Ester

concentration (Mm) reducing the germination index by50%

S-ABA 1 1.4 11R-ABA 2 1.4 4Analog 3 3.5 70Analog 4 >100 >100Analog 5 0.9 20Analog 6 >100 >100Analog 7 >100 11Analog 8 >100 10

Effect of the ABA Analogs on ABA-Responsive GeneExpression

To learn more about the components of the ABA recogni-tion response, the effects of the ABA analogs with biologicalactivity as germination inhibitors on individual ABA-respon-sive genes were determined. Effects of the ABA analogs onexpression of three ABA-responsive genes, dhn (rab), group 3lea, and Em, in embryos at 24 h after imbibition weredetermined (Fig. 3). Because analogs 7 and 8 had not provento be stable in solution, only compounds 1 through 6 were

0)(I)

01)

Er

U111111,_./ iis-P t 't

PM

Figure 2. Biological activity of (S)-ABA and analogs as germinationinhibitors. Structural formulas for the analogs are shown in Figure 1.Effects of concentration on the embryonic germination index areshown and data are the mean ± SD of three replicates. (S)-ABA (@),(R)-ABA (0), analog 3 (O), analog 4 (O), analog 5 (A), and analog 6(A).

cr-

1 4>+ ;

ABA Analogs

Figure 3. Comparison of the effects of (S)-ABA and the ABA analogsat a concentration of 10 MM on mRNA levels for the ABA-responsivegenes, dhn (rab), wheat /ea, and Em. Transcript levels were quantifiedby radioimaging and the relative intensity of the bands is shown.Experimental results are representative of five separate experiments.

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compared. Transcript levels were measured 24 h after imbi-bition because by that time control embryos show visible signsof germination, whereas ABA-treated embryos remaingrowth-arrested. Positive effects of(S)-ABA (1), (R)-ABA (2),and the (S)-series analogs 3 and 5 on dhn (rab) and wheat leatranscript levels were observed (Fig. 3), similar to the positiveactivities of these compounds as germination inhibitors. An-alogs 4 and 6 in the (R)-series with the saturated six-mem-bered ring that lacked biological activity as germination in-hibitors also had little effect on dhn (rab) and lea transcriptlevels. In contrast with the dhn and lea transcript results,induction of the Em gene by the ABA analogs did notcorrelate with their biological activity. Only (S)-ABA (struc-ture 1) and analog 5 were effective in inducing Em at 24 hpostimbibition. (R)-ABA (structure 2) and analog 3 were noteffective in inducing Em in the same time period, even thoughthese two compounds blocked embryonic germination effec-tively at 24 h.

In an extended time course study not shown, (R)-ABA(structure 2) was found to have little effect on Em levelsthrough 36 h postimbibition. Only after 48 h of imbibitiondid (R)-ABA (structure 2) have some positive effects on Emlevels, although by that time control embryos are in theadvanced stages of germination. At very high concentrations(100 ,uM), (R)-ABA is effective in inducing Em transcripts by24 h postimbibition. However, differences in the capability of(S)- and (R)-ABA to induce Em gene expression are stillobserved even at 100 ,M. The Em transcript level induced by100 Mm (R)-ABA is less than 50% of that induced by 100 Mm(S)-ABA.

Dose Response Effects of Analog 5 Comparedwith (S)-ABA on ABA-Responsive Gene ExpressionThe minimal concentrations of (S)-ABA and analog 5

required to induce the ABA-responsive genes were compared

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04

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Um

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dhn(rab)

lea (group 3)

Em

Figure 4. Dose response comparison of (S)-ABA and analog 5 forinduction of three ABA responsive genes, dhn (rab), wheat /ea, andEm. Typically, higher endogenous levels of the wheat lea transcriptwere present in the control water-treated sample compared withbackground levels for the other two transcripts.

600

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E

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400

200

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Figure 5. Endogenous (S)-ABA (pg/mg fresh weight) in embryosafter imbibition in 10 uM (S)-ABA (U), (R)-ABA (I), analog 3 (A),analog 5 (A), or water (0). (S)-ABA was measured by immunoassayas described in "Materials and Methods."

(Fig. 4). Concentrations of 1 Mm were partially effective and 4Mm were completely effective in inducing the ABA-responsivegenes. At 1 ,M, analog 5 induced transcript levels slightlyhigher than (S)-ABA. These results are very similar to theconcentration range of these two compounds found to beeffective in inhibiting germination (Fig. 2).

Effects of (R)-ABA and Analogs 3 and 5 on Endogenous(S)-ABA Concentration

It is not likely that the ABA analogs could be metabolizedto (S)-ABA, but it is possible that application of the analogscould indirectly affect endogenous (S)-ABA levels and sub-sequently induce gene expression. Possible effects of the ABAanalogs on endogenous (S)-ABA concentration were meas-ured (Fig. 5). The appropriate monoclonal antibodies specificfor (S)-ABA that do not cross-react with the analogs wereused for the immunoassays (28). Results of the (S)-ABAanalysis show that neither the application of (R)-ABA (2) oranalogs 3 and 5 had any effect on endogenous (S)-ABA levels.

DISCUSSION

Optically pure ABA analogs have been used to probe theshape of the ABA molecule required to elicit biological activ-ity. Effective germination inhibition ofdormant seed embryoswas obtained with (S)- and (R)-ABA, 1 and 2, and bothoptically active isomers of the methyl esters of acetylenicABA, 7a and 8a. The biological activity of both (S)- and (R)-ABA, 1 and 2, as well as (S)- and (R)-acetylenic methyl esters,7a and 8a, as germination inhibitors of wheat embryos isconsistent with the hypothesis that the 8'-methyl and the 7'-methyl can be transposed in the active site of the ABA

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receptor. Our results show that the dienoic acid side chain isnot rigorously required for recognition because analogs 7aand 8a, with more linear acetylenic side chains, were biolog-ically active. The pattern of activity observed for analogs 1through 4 is mirrored in compounds 5 through 8. In the caseof the dihydroABA analogs, only the optically active isomersof the (S)-ABA series 3 and 5 were effective. The change ofthe ring double bond to a single bond eliminated the biologicalactivity of the (R)-ABA series analogs 4 and 6 as germinationinhibitors. We conclude that the requirements for biologicalactivity must have more than a simple stereochemical com-ponent, and that the ring double bond plays a significant rolein the activity of the (R)-ABA series compounds. Studies toprobe this question are currently under way.An interesting result of our research is that biologically

active ABA analogs were identified as having differentialeffects on ABA-responsive gene expression (Fig. 3). All theanalogs that were effective germination inhibitors inducedtwo ABA-responsive genes, dhn (rab) and wheat lea, by 24 h,but those biologically active analogs had varying effects onEm induction. (S)-ABA (structure 1) and analog 5 wereeffective inducers of Em at a 10 uM concentration by 24 hpostimbibition. However, (R)-ABA (structure 2) and analog3, each effective germination inhibitors at the 1O-Mm concen-tration at 24 h, had little effect on Em mRNA levels by thattime. The stricter stereochemical requirement ofEm in dor-mant wheat seed embryo tissue is similar to the requirementofthe ABA receptor ofleaf stomata for (S)-ABA, but not (R)-ABA, to effect stomatal closure.A comparison of the effect of analog 3 with ABA on

expression of the Em gene in rice suspension cells has beenrecently conducted (23 and R. Quatrano, personal commu-nication). Results of that Em expression study show thatanalog 3 and ABA have different saturation kinetics in ricesuspension cells.The mechanism by which certain ABA analogs have differ-

ential effects on ABA-responsive gene expression is not yetknown. There may be more than one receptor involved inABA-responsive gene expression. Because (R)-ABA inductionof Em transcript in wheat embryonic tissue occurs slowly,there may be more steps involved in Em induction than forthe other ABA-responsive genes, and the additional steps maybe differentially affected by the ABA analogs. It is possiblethat (R)-ABA (structure 2) and analog 3 could be transportedmore slowly or metabolized more rapidly than the otherbiologically active ABA analogs. However, because (R)-ABA(structure 2) and analog 3 are just as active as (S)-ABA ininhibiting germination, these compounds must be transportedsufficiently into cells to effect germination inhibition. Anotherpossible explanation for differential gene expression is thatsome of the analogs are converted to (S)-ABA. That possibil-ity was examined by treating embryos with various ABAanalogs and then measuring endogenous (S)-ABA levels for24 h postimbibition (Fig. 5). No detectable increases in (S)-ABA levels were found after application of either (R)-ABA,analog 3, or analog 5.

In conclusion, our results demonstrate that optically pureABA analogs provide new tools to probe the ABA recognitionresponse. It appears that the ABA perception response that

results in the induction of Em has stricter stereochemicalrequirements than that for the group 3 lea and dhn (rab)genes.

ACKNOWLEDGMENTS

We acknowledge Angela C. Shaw and Nancy Lamb for synthesisof the analogs and Dr. Garth D. Abrams for helpful discussions.

LITERATURE CITED

1. Abrams SR, Milborrow BV (1991) Synthesis and biological ac-tivity of allenic analogs of abscisic acid. Phytochemistry 30:3 189-3 195

2. Abrams SR, Reaney MJT, Abrams GD, Mazurek T, Shaw AC,Gusta LV (1989) Ratio of (S)- to (R)-abscisic acid from plantcell cultures supplied with racemic ABA. Phytochemistry 28:2885-2889

3. Barthe P, Le Page-Degivry MT (1991) The resolution by HPLCof (R,S)-abscisic acid methyl ester and the metabolism of (-)-R- and (+)-S-abscisic acid by sunflower embryos. In Abstractsof the 14th International Conference on Plant Growth Sub-stances. Amsterdam, p 68

4. Close TJ, Kortt AA, Chandler PM (1989) A cDNA-based com-parison of dehydration-induced proteins (dehydrins) in barleyand corn. Plant Mol Biol 13: 814-821

5. Cummins WR, Sondheimer E (1973) Activity of the asymmetricisomers of abscisic acid in a rapid bioassay. Planta 111:365-369

6. Curry J, Morris CF, Walker-Simmons MK (1991) Sequenceanalysis of a cDNA encoding a group 3 LEA mRNA inducibleby ABA or dehydration stress in wheat. Plant Mol Biol 16:1073-1076

7. Dunstan DI, Abrams GD, Bock CA, Abrams SR (1992) Metab-olism of (+)- and (-)-abscisic acid by suspension cultures ofwhite spruce (Pices glauca) somatic embryos. Phytochemistry(in press)

8. Feinberg AP, Vogelstein B (1983) A technique for radiolabelingDNA restriction endonuclease fragments to high specific activ-ity. Anal Biochem 137: 266-267

9. Kienzle F, Mayer M, Minder ME, Thommen H (1978) Synthesisof optically active natural carotenoids and structurally relatedcompounds. III. Synthesis of (+)-abscisic acid, (-)-xanthoxin,(-)-loliolide, (-)-actinidiolide, and (-)-dihydroactinidiolide.Helv Chim Acta 61: 2616-2627

10. Lamb N, Abrams SR (1990) Synthesis of optically active cyclo-hexanone analogs of the plant hormone abscisic acid. Can JChem 68: 1151-1162

11. Litts JC, Greggory WC, Chakerian RL, Quatrano RS (1987) Thenucleotide sequence of a cDNA clone encoding the wheat Emprotein. Nucleic Acid Res 15: 3607-3618

12. Mertens R, Suning M, WeilerEW (1982) Metabolism oftritiatedenantiomers ofabscisic acid prepared by immunoaffinity chro-matography. Naturwissenschaften 69: 595-597

13. Milborrow BV (1986) The shape of abscisic acid and the activesite. In M Bopp, ed, Plant Growth Substances 1985. Springer-Verlag, Heidelberg, pp 108-119

14. Milborrow BV, Rubery PH (1985) The specificity of the carrier-mediated uptake of ABA by root segments of Phaseolus coc-cineus L. J Exp Bot 36: 807-822

15. Morris CF, Anderberg RJ, Goldmark PJ, Walker-Simmons MK(1991) Molecular cloning and expression of abscisic acid-responsive genes in embryos of dormant wheat seeds. PlantPhysiol 95: 814-821

16. Morris CF, Moffatt JM, Sears RG, Paulsen GM (1989) Seeddormancy and responses of caryopses, embryos, and calli toabscisic acid in wheat. Plant Physiol 90: 643-647

17. Mundy J, Chua N-H (1988) Abscisic acid and water-stress inducethe expression of a novel rice gene. EMBO J 7: 2279-2286

18. Ohkuma K (1965) Synthesis of some analogs of abscisic II. AgricBiol Chem 29: 962-964





19. Quarrie SA, Whitford PN, Appleford NEJ, Wang TL, HensonIE, Loveys BR (1988) A monoclonal antibody to (S)-abscisicacid: its characterisation and use in radioimmunoassay formeasuring abscisic acid in crude extracts of cereal and lupinleaves. Planta 173: 330-339

20. Railton ID (1987) Resolution of the enantiomers ofABA methylester using a stationary phase of cellulose tris (3,5-dimeth-ylphenyl carbamate)-coated silica gel. J Chromatogr 402:371-373

21. Rose PA, Abrams SR, Shaw AC (1992) Synthesis of chiralacetylenic analogs of the plant hormone abscisic acid. Tetra-hedron: Asymmetry 3: 443-450

22. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning,A Laboratory Manual, Ed 2. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, NY, pp 7.43-7.45

23. Schnall JA, Quatrano R (1991) The effect ofabscisic acid analogson the expression of the Em gene in rice suspension cells. InRB Hallick, ed, Abstracts, Third International Congress ofPlant Molecular Biology, Tucson, AZ, Oct 6-11, 1991, abstractNo. 880

24. Sondheimer E, Galson EC, Chang YP, Walton DC (1971) Asym-

metry, its importance to the action and metabolism of abscisicacid. Science 174: 829-831

25. Walker-Simmons M (1987) Embryonic ABA levels and sensitiv-ity in developing wheat embryos of sprouting resistant andsusceptible cultivars. Plant Physiol 84: 61-66

26. Walker-Simmons M (1988) Enhancement of ABA responsive-ness in wheat embryos by high temperature. Plant Cell Environ11: 769-775

27. Walker-Simmons MK, Abrams SR (1991) Use of ABA immu-noassays. In WJ Davies, HG Jones, eds, Abscisic Acid, Physi-ology and Biochemistry. Bios Scientific Publishers, London,pp 53-61

28. Walker-Simmons MK, Reaney MJT, Quarrie SA, Perata P,Vernieri P, Abrams SR (1991) Monoclonal antibody recogni-tion of abscisic acid analogs. Plant Physiol 95: 46-51

29. Walton DC (1983) Structure-activity relationships ofabscisic acidanalogs and metabolites. In FT Addicott, ed, Abscisic Acid.Praeger Press, New York, pp 113-146

30. Zeevaart JAD, Creelman RA (1988) Metabolism and physiologyof abscisic acid. Annu Rev Plant Physiol Mol Biol 39:439-473

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Optically Pure Abscisic Acid Analogs-Tools for - Plant Physiology

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