Proc. Nati. Acad. Sci. USAVol. 88, pp. 7266-7270, August 1991Plant Biology

Cis-acting DNA elements responsive to gibberellin and itsantagonist abscisic acid

(aleurone protoplast/gene expression/hormone response eement/lndudble enhnr)

KAREN SKRIVER*, FINN LOK OLSEN*, JOHN C. ROGERSt, AND JOHN MUNDY*f*Carlsberg Research Laboratory and Department of Physiology, Carlsberg Laboratory, Gl. Carlsberg Vej 10, DK-2500, Copenhagen, Denmark; and tDivisionof Hematology and Oncology, Washington University School of Medicine, St. Louis, MO 63110

Communicated by Diter von Wettstein, May 13, 1991

ABSTRACT We have used a transient expression assay inaleuroie protoplasts of barley to deite hormone responseelements of the absdisic acid (ABA)-responsive rice gene Rab-16A and of the gibberelln A3 (GA3)-resPOnve barley a-amy-lase gene Amy 1/6-4. Out approach used tracriptionalfusions between their 5' upstream sequences and a bacterialchloramphenil acetyltranderase reporter gene. A chimericpromoter containing six copies of the -181 to -171 region ofRab 16A fused to a minimal promoter conferred ABA-responsive expression on the reporter gene. Transcription fromthis ABA response element (GTACGTGGCGC) was unaf-fected by GA3. A chimeric promoter conta six copies ofthe-148 to -128 sequence of Amy 1/6-4 fused to them lpromoter conferred GA3-responsive expression on the reportergene. Transcription from this GA3 response dement (GGC-CGATAACAAACTCCGGCC) was repressed by ABA. Theeffect on transcription from both hormone response eentswas orientation-independent, indicating that they function asinducible enhancers in their native genes.

During late embryogenesis in cereals, abscisic acid (ABA)induces the expression of genes thought to mediate embryomaturation (1). Upon germination, gibberellin A3 (GA3) pro-motes the transcription of genes encoding a-amylase andother germinative enzymes in aleurone layers, a major site ofa-amylase synthesis (2, 3). In isolated aleurone cells, ABAinhibits GA3 effects on a-amylase transcription (3, 4). In vivo,this antagonism may mediate physiological events controllingthe switch from seed quiescence or dormancy to germination.To understand how ABA and GA3 regulate specific gene

expression and to elucidate their antagonism at the molecularlevel, Mundy et al. (5) have characterized an ABA-responsive rice gene, Rab 16A (rab-16A in ref. 5), and shownthat it is transcriptionally regulated by ABA. Genes homol-ogous to rice Rab 16A are highly conserved in maize andbarley and show the same patterns of expression in these andother species (6-8). Conserved 5' upstream sequences inthese genes have been identified as putative ABA responseelements (ABREs; refs. 5, 9, and 10). Recent studies on theABA-responsive Em gene of wheat have resulted in thecloning of a leucine-zipper protein that specifically binds tosuch a conserved sequence (11).Khursheed and Rogers (12) have characterized a GA3-

responsive barley a-amylase gene, Amy 1/6-4 (12). The 5'upstream region of this gene contains sequence motifs con-served among various GA3-responsive cereal genes (13, 14).These conserved sequences are present in a GA3-responsivewheat a-amylase promoter fragment (4), and they also maybe sites for protein binding in the promoter of a rice a-amy-

lase gene (15). Such conserved sequences are candidate GA3response elements (GAREs).We have used a transient expression assay in barley

aleurone protoplasts to further delineate the hormone re-sponse elements (HREs) of the Rab 16A and Amy 1/64genes. For this, we prepared transcriptional fusions betweentheir 5' upstream regions and reporter genes. Aleurone cellswere chosen because they respond to ABA and GA3 withchanges in specific gene expression (2, 3) and becauseexpression protocols have been developed for protoplastsderived from aleurone layers (16). Using promoters contain-ing tandemly repeated oligonucleotides covering conservedsequences of the Rab 16A and Amy 1/6-4 promoters fused toa minimal promoter containing the "TATA box" of thecauliflower mosaic virus 35S transcription unit, we show thatABA and GA3 regulate transcription of these genes viadistinct inducible enhancers. The mechanisms by whichthese enhancers may regulate target gene expression arediscussed.

METHODSReporter Gene Constrcs. All DNA manipulations were

performed by standard protocols (17). Plasmid DNAs used inthe expression assays were purified by CsCl gradient cen-trifugation, collected by ethanol precipitation, and resus-pended at 5 mg/ml in water. Various promoter constructswere fused to the Tn9 chloramphenicol acetyltransferase(CAT) coding region with a pea rbcS-E9 polyadenylylationsite (18). The major types of promoter constructs used wereas follows. (i) Long promoter (from nucleotide -941 to +9)and derivatives ofthe cauliflower mosaic virus 35S transcrip-tion unit. The 5' deletion derivatives at -90 (native EcoRVsite) and -46 (BAL-31 nuclease product) were constructedpreviously (19, 20). The latter is a minimal promoter con-taining only a functional TATA box. (i) Rab 16A and Amy1/64 promoters. A 5' upstream fragment of Rab 16A [-442(derived by BAL-31 digestion) to +27 (Nhe I)] and a 5'upstream fragment of Amy 1/64 [-639 (Xho 1) to +43(BamHI linker)] were cloned as blunt-ended fragments intothe blunt-ended HindIII site 5' to the CAT coding region (18).These promoters therefore contain homologous TATA boxesfrom the two hormone-responsive genes. (ii) Rab 16A andAmy 1/64 chimeric 35S promoters. These promoters weremade using oligonucleotides containing tandemly repeatedputative HREs. The oligonucleotides were synthesized toproduce cohesive BamHI ends upon annealing and were firstligated into theBamHI site in pUC12. Ofthe oligonucleotides

Abbreviations: ABA, abscisic acid; ABRE, ABA response element;CAT, chloramphenicol acetyltransferase; GA3, gibberellin A3;GARE, GA3 response element; HRE, hormone response element.*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"-in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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tested, four were shown to contain functional HREs:rab 2x -18CGACACCGTACGTGGCGCCACCGCCGCGCCT-158rab 6x -18GTACGTGGCGC-'71

amy lX -189CATCACTTGGGCATTGAATCGCCTTTTGAGCT-CACCGTACCGGCCGATAACAAACTCCGGCCGA-CATATC-'

amy6x -'1GGCCGATAACAAACTCCGGCC-11

Two kinds of chimeric promoters were constructed. In thefirst, the oligonucleotides were placed upstream of the 35Spromoter derivative deleted 5' to -46. To simplify cloningsteps, this basicTATA box/CAT gene construct was isolatedas a HindIII-Cla I fragment, and introduced into those sitesin pBluescript SK(-) (Stratagene). This produced a vectorwith numerous restriction sites 5' to the TATA box. Olignu-cleotides containing putative HREs were then subcloned asSma I-Pst I or Sma I-EcoRI fragments into the EcoRV/PstI or EcoRV/EcoRI sites 5' of the 35S TATA box. Thisproduced chimeric promoters with the oligonucleotides inboth orientations.

In the second kind of chimeric constructs, the oligonucle-otides were cloned as blunt-ended fragments into the EcoRVsite at -90 of the long 35S promoter. Sequences and restric-tion maps of all plasmids are available upon request.

Isolation of Barley Aleurone Protoplasts. Protoplasts wereisolated from aleurone cells by a method modified fromearlier protocols (2, 16, 21); the media used are described inTable 1. Initial experiments indicated that different seedstocks vary in their capacity to yield competent, viableprotoplasts. A stock (Hordeum vulgare cv. Himalaya, 1985harvest, Department of Agronomy, Washington State Uni-versity, Pullman, WA) was selected for this study that usuallyyielded 106 protoplasts per 50 seeds. Mature grains weredeembryonated and divided along the ventral groove. Aftersterilization (0.1% NaOCl, 20 min) and imbibition (sterilewater, 60 hr), aleurone layers with adhering testa/pericarpwere individually peeled from the starchy endosperm andtransferred to a 230-jm screen for cellulase predigestion (2hr) in APIM medium. The layers were then washed andtransferred on the screen to fresh enzyme solution anddigested for 16 hr. The washing and transfer steps ensuredthat released protoplasts were separated from endospermstarch, which otherwise causes detrimental increases in theosmotic strength of the incubation medium. Released proto-plasts were then purified on a 55% Percoll cushion in APTmedium.

Table 1. Media for protoplast experimentsComponent APIM APT PEG APM

Glucose, mM 109 109 109L-Arginine, mM 10 10CaCI2, mM 20 20Mes, mM 10 5 10Mannitol, mM 300 400 400 400MgCl2, mM 15Ca(NO3)2, mM 1000Gamborg B5 1x 1xPEG 3350 40%/Cellulase R-10 3%pH 5.4 5.6 9.0 5.4APIM, aleurone protoplast isolation medium; APT, aleurone pro-

toplast transfection medium; PEG, polyethylene glycol; APM, aleu-rone protoplast medium. All chemicals were from Sigma exceptpremixed Gamborg B5 (Flow Laboratories) and cellulase R-10(Seshin Pharmaceutical, Tokyo). pH was adjusted with HCI orKOH.Osmolarity of APIM, APT, and APM was 700 mosM. The PEGsolution was sterilized by autoclaving; the other media were steril-ized by filtration.

Protoplast Transfection and Transient CAT Expression As-says. Protoplasts (2 x 10i per sample) were transfected with50 ,ug of plasmid by PEG-mediated DNA uptake (21). Hor-mones (1 ,uM) were then added to the APM incubationmedium. Protoplasts were harvested 40 hr later, shown to bethe time of maximum CAT activity in initial experimentscomparing expression levels at days 1-5 after transfection.Following protein determinations with the Bradford reagent(Bio-Rad), CAT enzyme assays were performed at two ormore concentrations of extracted enzyme within the linearrange of the assay (<20%o acetylation). Activities were quan-titated by xylene extraction of the acetylated products (22)and were also monitored by thin-layer chromatography (23).Activity values given in Results represent the average of atleast three assays per plasmid construct.

RESULTSCAT Gene Expression from the 35S Promoter Is Not Sig-

nificantly Affected by ABA or GA3. The cauliflower mosaicvirus 35S promoter was used as a reference to optimize theexpression protocols and to examine the effect ofABA andGA3 on expression from this constitutive promoter (Fig. LA).While treatments with 1 ,uM ABA and GA3 slightly affectedoverall levels of protein synthesis in protoplasts, expressionof the 35S/CAT gene was not significantly affected by thehormones (Fig. 1B). These results are in agreement withthose from other studies (4, 24). This indicates that the CATtranscript produced in our experiments was relatively stablein cells treated with ABA and GA3. Thus, the effects of thehormones on CAT expression driven by the hormone-responsive promoters described below were mediated by 5'upstream sequences and resulted from differences in rates oftranscription.Rab 16A Promoter and Chimeric Derivatives Confer ABA-

Responsive Expression on CAT Reporter Genes. Initial exper-iments showed that expression of the CAT gene fused to theRab 16A promoter increased over basal levels upon treatmentwith ABA at 1 nM to 10 ,uM (data not shown). Subsequentexperiments were performed with 1 .tM ABA, which has

A

Bz

m00.

Em

-941 +9

-I35S PROMOTER

CON GA ABA GA/ABA

O- ES 9 0

w) Jia-

zw

00.

UL

FIG. 1. Effects of GA3 and ABA on transient expression of theCAT gene fused to the 35S promoter and on total protein accumu-lation in aleurone protoplasts. (A) Promoter ofthe cauliflower mosaicvirus 35S transcription unit (construct 1). (B) CAT enzyme assays ofprotoplasts transfected with the 35S promoter/CAT fusion constructincubated without (CON, control) or with GA3 (GA) and/or ABA (1,uM). Quantitation of CAT enzyme activities [units (U)/mg ofprotoplast protein] was by xylene extraction of products and deter-mination of total soluble protein extracted from protoplasts (mg persample, 2.5 x 10- protoplasts). One unit converts 1 nmol of chlor-amphenicol and acetyl-CoA to chloramphenicol 3-acetate and CoAper minute at pH 7.8 at 25°C. Bar patterns designating hormonetreatments are employed in subsequent figures.

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been used in previous studies (2, 5, 9). At this concentration,expression of the CAT gene fused to the Rab 16A promoterincreased 40-fold and was unaffected by treatment withequimolar GA3 (Fig. 2A, construct 2). Thus GA3 does notantagonize ABA-inducible gene expression under the timecourse and conditions used in these experiments. Recentstudies have indicated that conserved 5' upstream sequencesofRab and ABA-responsive genes from various species maybe ABREs (5, 9-11). In Rab 16A, these regions contain thesequence ACGTGGC (-179 to -173) and two copies of adegenerate sequence, CCGCCGCGCCT (-168 to -158) andCCGCCGCGCT (-130 to -121) that are within sites fornuclear protein binding.To determine whether these conserved sequences are

involved in transcriptional activation by ABA, oligonucleo-tides containing multiple copies ofthem were fused to the 35STATA box. Multiple copies of enhancer motifs have beenshown to boost inducible expression from various enhancers,presumably due to synergistic or combinatorial activation oftranscription by regulatory proteins (25). Expression of agene containing both conserved sequences was induced=40-fold by 1 ILM ABA (Fig. 2B, construct 3). This indicatedthat the ABRE(s) lies between -188 and -158 of Rab 16A.The control construct containing only the 35S TATA box(-46 to +9) did not produce detectable levels ofCAT enzymeactivity (data not shown).To confirm and further delineate the ABRE, an oligonu-

cleotide containing six copies of the sequence GTACGTG-GCGC (-181 to -171) was fused to the TATA box. Expres-sion ofthis gene was induced 6-fold following ABA treatmentand was unaffected by coincubation with GA3 (Fig. 2B,construct 4). In contrast, an oligonucleotide containing threecopies each of the -168 and -130 degenerate CCGC-CGCGC(C)T sequences was inactive (data not shown). Theoligonucleotide used in construct 4 promoted ABA-responsive expression in both orientations (data not shown),

A2

3

4

Bz

En

-442 +27| RAB PROMOTER |

-188 -158-46 35S +9I 2x RAB OLIGO I TATA

-181 -171 -46 35S +9I 6x RAB OLIGO I TATA

CON

ABA

BGA/ABA

FIG. 2. Effects ofGA3 and ABA on expression of the CAT genefused to 5' upstream sequences of Rab 16A. (A) Promoters con-structed from 5' upstream sequences of Rab 16A (construct 2) andoligonucleotides containing multiple, tandemly repeated copies ofRab 16A 5' upstream sequences fused to the heterologous 35S TATAbox (constructs 3 and 4). (B) Quantitation ofCAT enzyme activitiespromoted by these constructs. The construct containing only the 35STATA box (-46 to +9) did not produce detectable CAT enzymeactivity (data not shown).

indicating that the conserved ACGTGGC core sequencefunctioned as an ABA-inducible enhancer element.Amy 1/6-4 Promoter and Chimeric Derivatives Confer GA3-

Responsive, ABA-Repressible Expression on CAT ReporterGenes. Initial experiments showed that expression of theCAT gene fused to the Amy 1/64 promoter increased overbasal levels upon treatment with GA3 at 1 nM to 10 guM. Theeffect of 1 ,uM GA3 on CAT expression was examined insubsequent experiments. At this concentration, expressionof the CAT gene fused to the Amy 1/64 promoter increased8-fold over basal levels and was repressed by treatment withequimolar ABA (Fig. 3B, construct 5). This reporter genetherefore showed the pattern of transcriptional activation byGA3 and repression by ABA expected of a native a-amylasegene (2, 4). Several studies have noted conserved sequencesin the 5' upstream regions of a-amylase and other genes,which may be involved in their regulation by GA3 (13, 14). InAmy 1/64, the region between -200 and -100 contains twoof these conserved motifs whose core sequences are CTTTT(-167 to -163) and TAACAAA (-142 to -136).To determine whether these sequences are involved in

transcriptional regulation by GA3, an oligonucleotide (-189to -120) containing them was fused to the 35S TATA box(Fig. 3B, construct 6). This promoter showed the samepattern of hormone responsiveness as that with the -639 to+43 upstream fragment ofAmy 1/6-4. To determine whetherthe conserved sequence motifs within this region function asa GARE, oligonucleotides containing six copies of either-173 to -156 or -148 to -128 were fused to the 35S TATAbox. While the promoter containing the -167 CTTTT motifwas inactive (data not shown), expression from that contain-ing the -142 TAACAAA motif was induced 4-fold by GA3and was repressed by ABA (Fig. 3B, construct 7). This effect,seen for both orientations ofthe oligonucleotide (not shown),indicates that this sequence (GGCCGATAACAAACTCCG-GCC) functions as a GA3-inducible enhancer, or GARE.35S Promoters Containing the HREs Confer High-Level,

Regulated Expression on CAT Reporter Genes. To confirm theactivities of the HREs delineated above, chimeric promoterswith the ABRE or GARE oligonucleotides inserted at -90 ofthe long 35S promoter were tested (Fig. 4A). Such constructswould be expected to promote higher basal expression levels

A -6395E

-1896E

-14871

B 1200 Urz

0ELCD

E11.

+43AMY PROMOTER

-120-46 35S +9IITATA1 x AMY OLIGO

-127-46 35S +96x AMY OLIGO I TATA |

5 6 7

OCON

GA

GA/ABA

FIG. 3. Effects of the hormones on expression of the CAT genefused to 5' upstream sequences of Amy 1/64. (A) Promoter con-structs 5-7. (B) Quantitation ofCAT enzyme activities promoted bythese constructs.

FLF-L

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A 8

-941

-90 +9

-90 +91 domain B I domainJA

9 I 118bp 0x DNA I

10

11

C

z

0

oED

2000

1000

-181 -171

I 6x RAB OLIGO 1-148 -127

| 6x AMY OLIGO

OCON

ABA

GA

GA/ABA

10 11

FIG. 4. Effects of the hormones on expression of the CAT genefused to the 35S promoter and on derivatives with oligonucleotidescontaining multiple copies of the HREs. (A) Promoter constructs8-11. Construct 9 contained a 118-base-pair (bp) Hae III fragment ofthe replicative form of bacteriophage 4X174. (B) Quantitation ofCAT enzyme activities promoted by control constructs 1, 8, and 9.(C) CAT activities promoted by 35S chimeric promoters containingthe HRE constructs 10 and 11.

from the weak -90 to +8 promoter (5, 19), and perhapshigher inducible levels due to interactions between the HREsand activating elements within the long 35S promoter.

Expression from control promoters was initially examined(Fig. 4B, constructs 1, 8, and 9). A 5' deletion of the 35Spromoter to -90 reduced expression 5-fold compared withthe full promoter (Fig. 4B, constructs 1 vs. 8), in agreementwith results of earlier studies (5, 19). This indicated that the-90 to +9 region of the 35S promoter (domain A; ref. 19)functioned as a weak promoter. Insertion of random DNAfiagments (118 or 310 base pairs of phage 4X174 replicativeform) at the -90 EcoRV site slightly reduced expressionlevels compared with the long 35S promoter (Fig. 4B, con-structs 8 vs. 9). This indicated that insertions ofrandomDNAof approximately the same size as our oligonucleotides didnot radically alter expression from 35S promoters in whichthe upstream domain B is moved away from domain A (19).Expression from these control promoters (constructs 8 and 9)was not affected by hormone treatments (data not shown).

In contrast, the chimeric 35S promoters containing theHREs at -90 showed the expected patterns of hormone-responsive activation (Fig. 4C, constructs 10 and 11). CATexpression directed by the ABRE and GARE increased-12-fold in both cases following incubation with the respec-tive inducing hormones. As expected, GA3 induction fromthe chimeric promoter containing the GARE was repressedby coincubation with ABA (Fig. 4C, construct 11).Both of the chimeric promoters also directed the expres-

sion of higher levels of CAT activity in the absence orpresence of the hormones than promoters containing theoligonucleotides fused directly to the 35S TATA box (Fig.4C, constructs 10 and 11, vs. Fig. 2B, construct 4, and Fig.3B, construct 7). This is expected, as the chimeric 35S

promoters with the HREs at -90 contain additional activat-ing elements ofthe A and B domains. However, an importantfinding is that introduction of the HREs did not reduceexpression in the absence of the inducing hormones (Fig. 4B,construct 1, vs. Fig. 4C, constructs 10 and 11). This suggeststhat the ABRE and GARE do not repress transcription inthese promoters in the absence of their inducing hormones.

DISCUSSIONWe are analyzing the hormone-responsive expression of theRab 16A and Amy 1/64 genes as model systems to elucidatethe mechanisms ofaction ofthese hormones. Other work hasused deletion/mutation analyses with intact promoter se-quences to identify potential HREs (4, 5, 11). This approachmay measure the additive effects of multiple, different cis-acting elements that together influence transcription (19). Incontrast, our approach allows the study of the effects ofsingle, tandemly repeated elements, thereby simplifying thedefinition ofminimal sequences required to make a functionalHRE.Using this technique, we show here that multiple copies of

a conserved Rab 16A promoter sequence (rab 6x,181GTACGTGGCGC--171) confer ABA-responsive, tran-

sient expression on the CAT reporter gene in transfectedbarley aleurone protoplasts. These results are in agreementwith those of Guiltinan et al. (11), who showed that mutationof the sequence GGACACGTGGC to GGACcCGgGGC inthe promoter of the wheat Em gene abolished ABA-responsive expression of an Em promoter/f3-glucuronidasereporter gene. The same or closely related sequences arefound in other ABA-responsive genes and in genes known torespond to different signals (5, 11, 26).We also show here that multiple copies of an Amy 1/64

promoter sequence (amy 6x, -148GGCCGATAACAAAC-TCCGGCC'128) confers GA3-inducible, ABA-repressibleexpression on the CAT reporter gene. These results are inagreement with those of Huttly and Baulcombe (4), whoshowed that a 289-base-pair promoter region of a wheata-amylase gene containing a similar core sequence (GTAA-CAGAGTC) conferred similar patterns of hormone-responsive expression on a ,8-glucuronidase reporter gene intransfected oat aleurone protoplasts. The same or relatedcore sequences are found in the 5' upstream regions ofGA3-responsive genes including numerous a-amylases (14), athiol protease (-472GAACAAATTC-463; ref. 13), and a car-boxypeptidase (-108CAAGCAAAGTC -98, -222ATAA-CACGCT 213; ref. 27). Further work comparing GA3-responsive expression from these putative GAREs and mu-tants of the Amy 1/64 GARE identified here is needed todelineate the precise sequence requirements of functionalGAREs.To confirm these results and to initiate experiments to

elucidate the mechanisms by which the ABRE and GAREmay potentiate transcription, oligonucleotides containingthem were introduced into the 35S promoter at -90. Thenative 35S promoter consists of a mosaic of regulatoryelements whose overall effect is to confer constitutiveexpression on reporter genes (19). Thus, introduction ofHREs at -90 places them downstream of the B-domainenhancer region and upstream of the A domain, whichcontains at least one activating sequence and the TATA box.Such chimeric 35S promoters show the expected patterns ofhormone-responsive expression. In the absence ofhormonesthey promote higher expression levels than constructs con-taining only the HREs and the 35S TATA box. They alsodirect the same or higher expression levels than the long 35Spromoter. These results suggest that the ABRE and GAREdo not repress transcription in the absence of the hormones,a mechanism thought to regulate the expression of various

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other genes (28, 29). Instead, they appear to exert theircontrol over target gene expression primarily by activatingtranscription in response to their respective inducing hor-mones. However, due to the complexity ofthe 35S promoter,further study of the mechanisms by which the ABRE andGARE regulate transcription is required.Due to differences in the ABRE and GARE sequences, it

is unlikely that they are recognized by common regulatoryproteins, as has been noted for certain animal hormones (30).This suggests that the GARE and ABRE are recognized bydifferent regulatory protein factors. In the case of the Rabgene, this factor appears to be constitutively expressed at lowlevels because Rab mRNAs accumulate independently ofprotein synthesis (31). In vitro studies also indicate thatnuclear protein binding to the ABRE is only slightly inducedfollowing ABA treatments (5, 11). In contrast, GA3 inductionof a-amylase mRNA occurs after a lag phase requiringprotein synthesis, indicating that the factor(s) controlling thisresponse are newly synthesized (32). These earlier results,and those presented here, suggest that the antagonistic effectof ABA on GA-responsive transcription is primarily due todecreased activity, potentially controlled at the transcrip-tional, translational, and/or protein level, of the factor(s)interacting with the GARE.

We thank Suksawad Vongvisuttikun and Karina Arp for technicalassistance. F.L.O. was the recipient of a fellowship from the DanishCenter for Plant Biotechnology. This work was supported by EurekaGrant 270, Adaptation of Barley for Industrial Needs (ABIN). Thispaper is ABIN publication no. 43.

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