Proc. Natl. Acad. Sci. USAVol. 92, pp. 6640-6644, July 1995Plant Biology

The GA5 locus ofArabidopsis thaliana encodes a multifunctionalgibberellin 20-oxidase: Molecular cloning andfunctional expression

(vegetative tissue/2-oxoglutarate-dependent dioxygenase/cDNA construction/light-regulated expression/end-product repression)

YUN-LING Xu*, LI LI*t, KEQIANG Wu*, ANTON J. M. PEETERSt, DOUGLAS A. GAGE§, AND JAN A. D. ZEEVAART*¶*Michigan State University-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824; tDepartment of Genetics,Agricultural University, 6703 HA Wageningen, The Netherlands; and §Department of Biochemistry, Michigan State University, East Lansing, MI 48824

Communicated by Hans J. Kende, Michigan State University, East Lansing, MI, February 21, 1995 (received for review December 23, 1994)

ABSTRACT The biosynthesis of gibberellins (GAs) afterGA12-aldehyde involves a series of oxidative steps that lead tothe formation of bioactive GAs. Previously, a cDNA cloneencoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygenoxidoreductase (20-hydroxylating, oxidizing), EC 1.14.11.-]was isolated by immunoscreening a cDNA library from liquidendosperm of pumpkin (Cucurbita maxima L.) with antibodiesagainst partially purified GA 20-oxidase. Here, we reportisolation of a genomic clone for GA 20-oxidase from a genomiclibrary of the long-day species Arabidopsis thaliana Heynh.,strain Columbia, by using the pumpkin cDNA clone as aheterologous probe. This genomic clone contains a GA 20-oxidase gene that consists of three exons and two introns. Thethree exons are 1131-bp long and encode 377 amino acidresidues. A cDNA clone corresponding to the putative GA20-oxidase genomic sequence was constructed with the reversetranscription-PCR method, and the identity of the cDNAclone was confirmed by analyzing the capability of the fusionprotein expressed in Escherichia coli to convert GA53 to GA44and GA19 to GA20. The Arabidopsis GA 20-oxidase shares 55%identity and >80% similarity with the pumpkin GA 20-oxidaseat the derived amino acid level. Both GA 20-oxidases sharehigh homology with other 2-oxoglutarate-dependent dioxyge-nases (2-ODDs), but the highest homology was found betweenthe two GA 20-oxidases. Mapping results indicated tightlinkage between the cloned GA 20-oxidase and the GAS locusof Arabidopsis. The ga5 semidwarf mutant contains a G Apoint mutation that inserts a translational stop codon in theprotein-coding sequence, thus confirming that the GAS locusencodes GA 20-oxidase. Expression of the GAS gene in Ara-bidopsis leaves was enhanced after plants were transferredfrom short to long days; it was reduced by GA4 treatment,suggesting end-product repression in the GA biosyntheticpathway.

The gibberellins (GAs) are tetracylic diterpenoid compoundsthat play an important role in many aspects of plant growth anddevelopment, such as promotion of cell division and extension,seed germination, stem growth, and fruit set (1). The biosyn-thesis of GAs has been studied extensively in developing seeds(2, 3), but their function in immature seeds, if any, is unknown.In contrast, relatively little is known about GA biosynthesis invegetative tissues, because GA levels in these tissues are lowcompared with levels in immature seeds, and active enzymepreparations for GA conversions are difficult to obtain. It is,therefore, desirable to extend studies on regulation of GAbiosynthesis to green plants in which GAs have definite roles.One of these roles is to mediate photoperiodic control of stemelongation in rosette plants (4-6). Therefore, to understand

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6640

the mechanism by which long days (LD) cause stem elongation,it is necessary to elucidate how daylength regulates GAbiosynthesis at the biochemical and molecular levels.

In Arabidopsis thaliana, a long-day plant (LDP) suitable formolecular genetic studies (7), a number of GA-responsivedwarf mutants have been isolated (8). It has been demon-strated that one of these mutants carrying thegaS mutation hasreduced levels of Cl-GAs, indicating that the product of theGAS gene catalyzes elimination of C-20 at the aldehyde levelin the GA biosynthetic pathway. ThegaS dwarf mutant also hasincreased levels of certain C20-GAs, indicating existence of anadditional control, possibly hydroxylation of C-20 (9). Thissuggests that the GAS locus encodes a GA 20-oxidase [gib-berellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxy-lating, oxidizing), EC 1.14.11.-]. In pumpkin endosperm, theenzyme activities for the steps between GA53 and GA20 (Fig.1) reside on a single polypeptide (10), indicating that GA20-oxidase is a multifunctional enzyme that catalyzes theoxidation and elimination of C-20 and thus plays a pivotal rolein the conversion of C20- to C19-GAs. Recently, cDNA clonesencoding GA 20-oxidases that differ by one amino acid havebeen isolated from developing cotyledons (11) and liquidendosperm (12) of developing pumpkin seeds. We report hereon the molecular cloning and functional expression of a GA20-oxidase from Arabidopsis, using the GA 20-oxidase cDNAfrom pumpkin endosperm as a heterologous probe, and showthat it is encoded by the GAS locus.11 Furthermore, we showthat expression of this gene is enhanced by transfer of plantsfrom short-day (SD) to LD conditions and is reduced bytreatment with an active GA.

MATERIALS AND METHODSPlant Material. Plants of Arabidopsis thaliana Heynh. (L.),

strain Landsberg erecta (Ler) or Columbia (Col), were grownas described (9). When required, plants in the rosette stagewere sprayed with an aqueous solution of 30 ,uM GA4,containing 5% (vol/vol) ethanol and 0.05% Tween 20.The two mutant lines Ler (cer2/cer2,gaS/ga5,EMB20/

EMB20) and Col (CER2/CER2,GAS/GA5,emb20/emb20) werecrossed, and the recombinants between GAS and the flankingmarkers CER2 and EMB20 were used for restriction fragment-length polymorphism mapping. For each recombinant, an F3

Abbreviations: Col, Columbia; GA, gibberellin; LD, long day(s); LDP,long-day plant; Ler, Landsberg erecta; 2-ODD, 2-oxoglutarate-dependent dioxygenase; RPA, RNase protection assay; SD, shortday(s); YAC, yeast artificial chromosome.tPresent address: Department of Plant Breeding, Cornell University,Ithaca, NY 14853.To whom reprint requests should be addressed.1The sequences reported in this paper have been deposited in theGenBank data base [accession nos. U20872 (Ler), U20873 (Col), andU20901 (ga5/Ler)].

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CHO OHC02H ..-Oc H/ H

COH C02H GA19 H C02H GA17

+ C C20~~~+0H CO2H GA20

FIG. 1. Reactions catalyzed by GA 20-oxidase in the early-13-hydroxylation pathway.

progeny was grown to score the genotype of the correspondingF2 plant and to prepare DNA for mapping.

Nuclekc Acid Isolation. Total genomic DNA from Arabidop-sis leaves was extracted and purified as described (13, 14).DNA from yeast artificial chromosomes (YACs) was extractedas described by Gibson and Somerville (15).

Total RNA was extracted from Arabidopsis leaves by usingan Extract-A-Plant RNA isolation kit (Clontech). PlasmidDNA was extracted as described by Sambrook et al. (16). Forsequencing, the DNA was purified with a Wizard miniprepsDNA purification system (Promega).

Southern Blot Analysis and Library Screening. DNA sam-ples digested with various restriction enzymes were separatedby electrophoresis in 0.8% agarose gels in 0.089 M Trisborate/0.025 M EDTA buffer and transferred to Hybond+(Amersham) membranes by capillary blotting with 0.4 MNaOH. Baked blots were prehybridized and hybridized in 0.5M Na2HPO4, pH 7.2/7% SDS/1 mM EDTA at 68°C andwashed in 40 mM Na2HPO4, pH 7.2/5% SDS/1 mM EDTA at68°C. The hybridization probes were radiolabeled with[32P]dCTP by random priming with a Prime-A-Gene labelingsystem (Promega).An Arabidopsis genomic library of Col DNA constructed in

AGEM11 (provided by R. W. Davis, Stanford University) wasscreened by plaque hybridization. The filters were hybridizedwith the 32P-labeled pP16 insert (a pumpkin GA 20-oxidasecDNA clone; ref. 12) and washed at 46°C in solutions asdescribed for Southern blot analysis.DNA Sequencing, Sequence Analysis, and Oligonucleotide

Synthesis. Dye primer sequencing of cDNA or genomic clone(or subclone) inserts in pBluescript SK(+) (Stratagene) anddye terminator sequencing of PCR products were performedwith a 373A sequencer (Applied Biosystems). DNASIS was usedto compare the DNA sequences and to deduce the proteinsequences from the nucleotide sequences. Protein sequencealignment was performed by using PROSIS (Hitachi SoftwareEngineering).

Oligonucleotides were synthesized by phosphoramiditechemistry with a 394 DNA/RNA synthesizer (Applied Bio-

systems). The oligonucleotides synthesized were JZ7 (5'-ACATGG TCT TGG TGA AGG AT-3'), JZ8 (5'-CCA AGC TTCCAT GGA AGG A-3'), JZ10 (5'-TTC TTA GAT GGG TTTGGT GA-3'), and JZ11 (5'-AAA ATG GCC GTA AGT TTCGT-3').

Reverse Transcription-PCR and Construction of a PutativecDNA Clone forArabidopsis GA 20-Oxidase. A total of 1 ,ug ofRNA (isolated as described above) was used with a lst-STRANDcDNA synthesis kit (Clontech) to synthesize first-strand cDNAin a volume of 20 ,tl. Four microliters of first-strand cDNA wasamplified in a 50-j.d volume by using a GeneAmp PCR reagentkit with AmpliTaq polymerase (Perkin-Elmer) and 2.5 ,tMJZ8 and JZ10 primers. The cDNA template was initiallymelted at 94°C for 5 min and then subjected to 40 amplificationcycles of 1 min of denaturing at 95°C, 2 min of annealing at48°C, and 2 min of extension at 72°C. A final extension wasperformed at 72°C for 7 min. The HindlIl-digested PCRproducts were cloned into the pBluescript SK(+) EcoRV/HindlIl site, and the fragment corresponding to the 3' end ofthe A17-4 clone without the introns was named pH30 (Fig. 2).

Twenty-five nanograms of A17-4 DNA was amplified asabove with JZ7 and JZ1 1 as primers. The product was digestedwith Hindlll, and the 5' end was cloned into the pBluescriptSK(+) EcoRV/HindIII site and named pS20 (Fig. 2).The pH30 clone was digested with Sma I and Hindlll, and

the insert was subcloned into the HindIII/HinclI site of pS20and named pH3020, giving a full-length cDNA clone thatcorresponded to the putative GA 20-oxidase (Fig. 2).

Preparation ofE. coli Protein Extracts and Enzyme Assays.E. coli XL1-Blue cells containing pH3020 were grown to anOD600 of 0.5. Isopropyl ,B-D-thiogalactoside was then added togive a concentration of 0.5 mM, and the cells were grown foranother 2 h. After centrifugation, the cells were resuspendedin lysis buffer (50mM Tris HCl, pH 8.0/3 mM dithiothreitol/1mg of lysozyme per ml) and incubated at room temperature for10 min. After centrifugation, the supernatants were concen-trated against polyethylene glycol and used to assay oxidationof GA53 and GA19 as described (17), except that the 2-oxo-glutarate concentration was adjusted to 25 mM. The substrates[14C]GA53 (4.1 GBq/,umol) and [14C]GA19 (4.8 GBq/,umol)were prepared as described (17). The products of enzymeassays were separated from the substrates with a ABondapakC18 column (30 x 0.4 cm). A 30-min gradient of 20-80%(vol/vol) methanol in 1% aqueous acetic acid at a flow rate of2.0 ml/min was used. The effluent from the HPLC column was

12.3 kb , 4.0 kb I

5.0 kb ,1.9 kb ,1.9 kb,Xhol Xhol Xho EoRI Xhol Xhol

- ;_7-4EX pA17-4S2EcoRI

Jz11~ JZ8 %

ATG Exon

A17-4 as templateJZ7 and JZ11 as primersPCR product digested witt

'IpS20 ATG HIn

ATG

Xhol

JZ7

Xhol

,JZ10

Intron Exon Intron Exon TAA

1st-strand cDNA as templateJZ8 and JZ1O as primers

h Hlndil PCR product digested with Hlndil

di Hln j pH30dl1l Hindill TAA

-AA pH3020TAAHIndlil

FIG. 2. Restriction map of A17-4 clone and its subclones (Upper)and diagram of construction of a cDNA clone for GA 20-oxidase ofArabidopsis (Lower).

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6642 Plant Biology: Xu et al.

passed through a radioactive flow detector (Flo-One model;Radiomatic, Meriden, CT) using a 250-,ul solid scintillator flowcell. Radioactive peaks were collected, dried, methylated, andagain purified by reverse-phase HPLC with a 30-min gradientof 30-90% methanol in water at 2.0 ml/min. The radioactivefractions were dried and trimethylsilylated. The samples were

analyzed by combined GC/MS as before (6).mRNA Quantitation. GA5 mRNA levels were quantified by

RNase protection assay (RPA) with an RPA II kit (Ambion,Austin, TX). RPA was performed according to the manufac-turer's instructions, except that a phosphor screen (MolecularDynamics) was exposed to the gel. Each experiment was

repeated three times with similar results.

RESULTS AND DISCUSSION

Isolation and Sequence Analysis of Arabidopsis GenomicClones Encoding GA 20-Oxidase. With a pumpkin GA 20-oxidase cDNA clone (pP16; ref. 12) as a probe, 1.5 x 105recombinant phage plaques from an Arabidopsis genomiclibrary were screened. A total of 15 positive clones was

recovered from the library. One of these clones, A17-4, was

further analyzed.The A17-4 clone was digested with Xho I, and the resulting

smallest fragment (Fig. 2), which is about 1.9 kb long and ableto hybridize with the pP16 probe, was subcloned (designatedas pA17-4S2) and sequenced. The results indicated that thepAl7-4S2 clone contains an Arabidopsis homolog of GA20-oxidase of pumpkin. However, the pA17-4S2 clone did notinclude the entire sequence of the putative GA 20-oxidasegene. Thus, the adjacent EcoRI/Xho I fragment (Fig. 2) fromA17-4 was subcloned (designated pA17-4EX) and sequenced.These two subclones spanned the entire putative GA 20-oxidase sequence of Arabidopsis. Another clone, A9-2, waspartially sequenced and had exactly the same sequence as thatof pA17-4S2.Comparison of the putative Arabidopsis GA 20-oxidase

genomic sequence with the pumpkin GA 20-oxidase cDNAclone sequence showed that the Arabidopsis gene containsthree exons and two introns (Fig. 3). The three exons are 1131bp long and encode a putative protein of 377 amino acidresidues of Mr 43,381. The first intron occurs at base pair 551from the starting codon and is 191 bp long, whereas the secondis located at base pair 873 from the starting codon and is 103bp long. Both introns possess the consensus 5' GT splice donorsite and the 3' AG splice receptor site (Fig. 3). Both intronsalso contain >65% A+T, so that they can be accurately andefficiently spliced in vivo (18).The DNA sequence of the putative Arabidopsis GA 20-

oxidase shares 60% identity with that of the pumpkin GA20-oxidase. At the derived amino acid level, there is 55%identity and >80% similarity between the putative Arabidopsisand the pumpkin GA 20-oxidases.GA 20-oxidase is a member of the 2-ODD family of proteins.

The amino acid sequences deduced from Arabidopsis genomicDNA and the pumpkin cDNA clone share high homology withthe functional domains of several other 2-ODDs (11, 19, 20).The proposed consensus sequence Asn-Tyr-Tyr-Pro-Xaa-Cys-Xaa-Xaa-Pro (positions 230-238; Fig. 3) of 2-ODDs forbinding the common cosubstrate, 2-oxoglutarate, and the threehistidine residues (His-98, -247, and -303) for binding Fe2+ are

all conserved in the putative GA 20-oxidase. However, thehighest homology was found between the putative ArabidopsisGA 20-oxidase and the pumpkin GA 20-oxidase. We proposethat the sequence Leu-Pro-Trp-Lys-Glu-Thr (positions 148-153; Fig. 3), which is conserved in the GA 20-oxidases ofpumpkin (11) and Arabidopsis (Fig. 3), may be involved in thebinding of the GA substrate because this motif is highlydiversified in other 2-ODDs.

Proc. Natl. Acad. Sci. USA 92 (1995)

Jzll B

1 ATG GCC GTA AGT TTC GTA AGA ACA TCT CCT GAG GAA GAA GAC AAA1 M A V S F V R T S P E E E D K

46 CCG AAG CTA GGC CTT GGA AAT ATT CAA ACT CCG TTA ATC TTC AAC16 P K L G L G N I Q T P L I F N

91 CCT TCA ATG CTT GAC CTT CAA GCC AAT ATG GCA AAC CAA TTT CAC31 P 8 N L D L Q A N N A N Q F H

136 TGG CCT GAC GAC GAA AAA CCT TCC ACT TTG CAA CTC GAG CTT GAT46 w P D D E K P s T L Q L E L D

181 GTT CCT CTC ATC GAC CTT CAA AAC CTT CTC TCT GAT CCA TCC TCC61 V P L I D L Q N L L S D P S S

226 ACT TTA GAT GCT TCG AGA CTG ATC TCT GAG GCC TGT AAG AAG CAC76 T L D A B R L I S E A C K K H

271 GGT TTC TTC CTC GTG GTC AAT CAC GGC ATC AGC GAG GAG CTT ATT91 G F F L V V N H G I S E E L I

316 TCA GAC GCT CAT GAA TAC ACG AGC CGC TTC TTT GAT ATG CCT CTC106 S D A H E Y T 8 R F F D N P L

361 TCC GAA AAA CAG AGG GTT CTT AGA AAA TCC GGT GAG AGT GTT GGC121 S E K Q R V L R K S G E S V G

HindIII406 TAC GCA AGC AGT TTC ACC GGA CGC TTC TCC ACC AAG CTT CCA TGG136 Y A S S F T G R F S T K T P-

Jz8451 AAG GAG ACC CTT TCT TTC CGG TTT TGC GAC GAC ATG AGC CGC TCA151 I U P-T L S F R F C D D M S R S

496 AAA TCC GTT CAA GAT TAC TTC TGC GAT GCG TTG GGA CAT GGG TTT166 K S V Q D Y F C D A L G H G F

GTAAAAGTTAAAACAGAGCAAATAATCTAAAAAAGAAAGAGATAGAGAAATCAAACATTTAACATGTCTTTTGTCTTTTCCTCTGTTTTTTTTATATAGAAAACGTCTTAAACCAATAACCGGTTTCTTCTATACCGACCACTGTACCGTAACTGTAGAAGACTTATGTTAATGTTGGTTTGTGGTTGCAG

541 CAG CCA TTT GGG AAG GTG TAT CAA GAG TAT TGT GAA GCA ATG AGT181 Q P F G K V Y Q E Y C E A M S

586 TCT CTA TCA CTG AAG ATC ATG GAG CTT CTG GGG CTA AGT TTA GGC196 S L S L K I M E L L G L S L G

631 GTA AAA CGG GAC TAC TTT AGA GAG TTT TTC GAA GAA AAC GAT TCA211 V K R D Y F R E F F E E N D S

676 ATA ATG AGA CTG AAT TAC TAC CCT CCA TGT ATA AAA CCA GAT CTC226 I N R L N V P L

721 ACA CTA GGA ACA GGA CCT CAT TGT GAT CCA ACA TCT CTT ACC ATC241 T L G T G P H C D P T S L T I

JZ7766 CTT CAC CAA GAC CAT GTT AAT GGC CTT CAA GTC TTT GTG GAA AAT256 L H Q D H V N G L Q V F V E N

A (ga5)811 CAA TGG CGC TCC ATT CGT CCC AAC CCC AAG GCC TTT GTG GTC AAT271 Q W R S I R P N P K A F V V N

GTAACCCATTTAGATTTTTAAAAACCTGCACATCTTGGTTAGATATATACAGCTTTGGCTAGGTACCTCCATATACTAAACTGGGCTTAATATTGTTTTGTAG

856 ATC GGC GAT ACT TTC ATGOGCT CTA TCG AAC GAT AGA TAC AAG AGC286 I G D T F X A L B N D R Y K 8

901 TGC TTG CAC CGG GCG GTG GTG AAC AGC GAG AGG ATG AGG AAA TCA301 C L H R A V V N S E R M R K S

946 CTT GCA TTC TTC TTG TGT CCG AAA AAA GAC AGA GTA GTG ACG CCA316 L A F F L C P K K D R V V T P

991 CCG AGA GAG CTT TTG GAC AGC ATC ACA TCA AGA AGA TAC CCT GAC331 P R 1 L L D S I T S R R Y P D

1036 TTC ACA TGG TCT ATG TTC CTT GAG TTC ACT CAG AAA CAT TAT AGA346 F T w S N F L E F T Q K H Y R

AJZ101081 GCA GAC ATG AAC ACT CTC CAA GCC TTT TCA GAT TGG CTC ACC AAA361 A D N N T L Q A F S D W L T K

1126 CCC ATC TAA376 P I *

FIG. 3. Genomic DNA sequence of an Arabidopsis (Col) GA20-oxidase gene and the deduced amino acid sequence. The nonnum-bered sequences represent two introns, and the arrowheads indicatethe positions of the introns between positions 551 and 552 and between873 and 874. Shaded residues are highly conserved in 2-ODDs, exceptfor the sequence Leu-Pro-Trp-Lys-Glu-Thr, which is conserved only inGA 20-oxidases (see text). At the amino acid level, Col has 97.1%identity and 98.7% similarity with Ler. The point mutation G -* A atnucleotide 816 in gaS is indicated.

Functional Expression of the GA 20-Oxidase cDNA Clone inE. coli. A cDNA clone for the putative GA 20-oxidase wasconstructed as described in Materials and Methods and desig-

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nated pH3020. The cDNA sequence completely matched thecorresponding genomic sequence, but without the two introns(Fig. 2). To confirm the identity of the pH3020 clone, theencoded protein was expressed as a fusion protein in E. coliXL1-Blue, and the capability of the expressed protein tometabolize the substrates [14C]GA53 and [14C]GA19 was de-termined. GA 20-oxidase activity was detected in proteinextracts from E. coli XL1-Blue containing pBluescript SK(+)with the cDNA insert in frame with the ,3-galactosidase openreading frame of pBluescript (Fig. 4 B and D), but no activitywas detected in protein extracts from E. coli XL1-Blue con-taining pBluescript with the cDNA insert out of frame with the13-galactosidase reading frame (Fig. 4A and C). [14C]GA53 waspredominantly converted to [14C]GA44 with small amounts ofradioactivity eluted at the HPLC retention time of [14C]GA19(Fig. 4B), whereas [14C]GAIg was converted to [14C]GA20 (Fig.4D). The products [14C]GA44 and [14C]GA2o were identified byfull-scan GC/MS; insufficient material of the putative['4C]GA19 product (Fig. 4B) was available for identification byGC/MS. Thus, a single enzyme catalyzes two consecutive

20 25 30 35 20 22 24 26

oxidations at C-20 during GA biosynthesis. Furthermore, theenzyme also catalyzes the loss of C-20 from the aldehydeprecursor and is therefore responsible for each step in theconversion of C20-GAs to C19-GAs (Fig. 1). The same obser-vation was made with the recombinant enzymes from pumpkincotyledons (11) and endosperm (12). However, a difference incatalytic properties was observed between the Arabidopsis andpumpkin recombinant enzymes. In the reaction catalyzed bythe pumpkin recombinant enzyme, GA19 was converted mostlyto GA17 (11), while in the reaction catalyzed by theArabidopsisrecombinant enzyme, GA19 was converted to GA20 only-theimmediate precursor of bioactive GA1. This correlates with theobservation thatArabidopsis produces predominantly C19-GAs(9), whereas developing pumpkin seeds produce biologicallyinactive C20-tricarboxylic acids as major products (11). Fe2 ,2-oxoglutarate, and ascorbate were required as cofactors forthe conversions of GAs by the fusion protein (data not shown),thus confirming that the GA 20-oxidase ofArabidopsis belongsto the 2-ODD family of enzymes (11, 19, 20).The GA5 Locus of Arabidopsis Encodes the GA 20-Oxidase

Harbored in the A17-4 Clone. Southern hybridization ofgenomic DNA from Col used pAl7-4S2 as a probe. Twelveenzymes were used to digest genomic DNA of which Xho I,EcoRI,Xba I, BamHI, Sac I, Cla I, Sca I, and Sal I gave a singleband, and Bgl II, Hindlll, EcoRV, and Dra I gave two bands(data not shown). The latter four restriction sites were foundin the pAl7-4S2 clone. It is likely, therefore, that pAl7-4S2represents a single copy sequence in the Arabidopsis genome.The GA5 locus is located on chromosome 4 and is flanked

by CER2 and EMB20. The map distance from GA5 to CER2and EMB20 is about 0.5 and 2.0 centimorgans (cM), respec-tively (ref. 21; Fig. 5). Recombinants between GAS and CER2and between GAS and EMB20 (see Materials and Methods)were selected for restriction fragmentation-length polymor-phism screening. Of eight recombinants, including four re-combinants between GAS and CER2 and four recombinantsbetween GAS and EMB20, no recombination was found be-tween pA17-4S2 and GAS. Thus, the distance between pA17-4S2 and GAS is .0.1 cM (19 kb 5.2 x 10-6 cM per bp; ref.23) from the CER2 side and .0.4 cM (77 kb) from the EMB20side. This indicates that the gene corresponding to pAl 7-4S2is very closely linked to the GAS locus, if not identical.For cloning the ABI] gene, chromosome walking was per-

formed in the region spanning the GAS locus (Fig. 5; ref. 22).DNAs from YACs EG2A5 and EGlF12 (prepared from Colgenomic DNA), which putatively span the GAS locus, werehybridized with pAl7-4S2 and showed the same band patternsas Col genomic DNA, but DNA from YAC EW3H7 did nothybridize with pA17-4S2 (data not shown). To further dem-onstrate that YACs EG2A5 and EG11I2 contain the pAl7-4S2 sequence, a fragment of each YAC was amplified by PCRwith JZ7 and JZ8 as primers. The amplified fragments weresequenced and showed exactly the same sequence as thecorresponding fragment of pAl7-4S2.To confirm that the Al7-4 clone (obtained from Col DNA)

represents the GA5 locus, PCR was performed on genomicDNA from gaS and wild type (Ler) plants with the JZI I and

Retention time, min

FIG. 4. HPLC analysis of incubations of [14C]GA53 (A and B) and[14C]GAi9 (C and D) with protein extracts from E. coli that expresspH3020 either out of frame (A and C) or in frame (B and D) with the,B-galactosidase open reading frame of pBluescript. Substrates andproducts were identified by GC/MS. m/z (% relative abundance) forGA53: 456(44), 448(57), 424(11), 416(16), 395(17), 389(29), 209(100),and 207(82); for GA44: 440(46), 432(74), 379(12), 373(18), 240(29),238(38), 209(76), and 207(100); for GAi1: 442(67), 434(74), 410(19),402(21), 381(22), 380(26), 375(28), and 374(31); and for GA20:426(64), 418(100), 381(28), 375(44), 365(8), 363(15), 307(9), 301(13),209(17), and 207(17).

cer2 ga5

51.9 52.4

100 kb

emb2O abil

5EG2A5

54.4 55.0

EW3H7

FIG. 5. Summary of mapping anArabidopsis GA 20-oxidase to theGAS locus. The genetic positions of cer2, gaS, emb2O, and abil on

chromosome 4 are indicated as described (21). Two YACs are shownrepresenting the corresponding Arabidopsis genomic region (22).

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i i il

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JZ10 as primers. The amplified fragments were sequenced anda G -* A mutation, resulting in a premature stop codon, was

found in the GA 20-oxidase gene of the gaS mutant at position816 (Fig. 3). It is expected that this mutation results in thesynthesis of a truncated polypeptide (271 instead of 377 aminoacids) with either diminished or complete loss of catalyticactivity (null mutant). Thus, a severe dwarf phenotype as in thegal, ga2, and ga3 mutants (8) would be expected. However, gaSplants are semidwarf and produce C19-GAs, albeit in reducedquantities (9). This suggests that the function of the GA5product is supplemented by an additional GA 20-oxidaseactivity. Indeed, three GA 20-oxidase cDNA clones have beenisolated from Arabidopsis, one of which corresponds to thetranscript of the GA5 locus reported here (P. Hedden, personalcommunication).

Regulation of GA5 Expression in Arabidopsis Leaves byLight and GA. When plants were transferred from SD to LDconditions, GA5 mRNA in leaves increased about 40%. Nosignificant change in the amount of GA5 mRNA was detectedwhen the plants in the LD condition were transferred todarkness for 24 hr (Fig. 6). Since the Ler strain of Arabidopsiswill also bolt under SD conditions (quantitative LDP), albeitdelayed in comparison with plants under LD conditions, therelatively small increase in GA5 mRNA under LD conditions(Fig. 6) is not unexpected. It will be of interest to investigatethe regulation of GA 20-oxidase by daylength in other species,such as spinach, that are qualitative LDPs (6).The level of GA5 mRNA was higher in the gaS and gai

(GA-insensitive) mutant than in wild-type Arabidopsis leaves.Treating the plants with GA4 (a down-stream product of GA20-oxidase), caused GA5 mRNA levels to decrease in leaves ofwild-type and gaS mutant plants, while the level increasedslightly in gai mutant leaves (Fig. 6). Down-regulation of GA20-oxidase mRNA by GA3 has also been observed in the galmutant of Arabidopsis (ref. 24; P. Hedden, personal commu-

nication). These results support the idea that growth inducedby GA exerts a feedback effect on GA biosynthesis (25, 26).Mutant gai plants accumulate bioactive GAs to a higher levelthan do wild-type plants (27) and do not respond to appliedGA (28). It is of interest, therefore, that gai plants also had a

higher level of GA5 mRNA compared with wild-type plants.But in contrast to wild-type and gaS plants, GA5 mRNA didnot decrease in GA-treated gai plants (Fig. 6). This suggests

S L D WT ga5 gai

FIG. 6. Expression of GAS gene in Arabidopsis leaves as shown byimages of GA 20-oxidase mRNA obtained by RPA. The antisenseRNA of GA 20-oxidase from base pair 444 to base pair 1 (Fig. 3) wasradiolabeled with [32P]CTP by using T7 RNA polymerase. (UpperLeft)SD-grown plants (lane S) were transferred to LD conditions for 5 days(lane L) and then were moved to darkness for 24 h (lane D). Relativeintensities were 100 (lane S), 143 (lane L), and 157 (lane D). For eachassay, 100 ,ug of RNA was used. (Upper Right) Effect of applied GA4.The plants were treated with GA4, and the leaves were harvested 24hr later. Lanes: +, GA4 applied; -, no GA4 applied. Relativeintensities for wild type were 100 (lane -) and 75 (lane +), forgaS were165 (lane -) and 78 (lane +), and for gai were 165 (lane -) and 200(lane +). For each assay, 80 Zg of RNA was used. (Lower) Ethidiumbromide-stained agarose gels containing 30 ,ug of RNA of the corre-

sponding samples used for the RPAs.

that the GAI product plays a role in the regulation of GAbiosynthesis.

In conclusion, by using a cDNA clone for GA 20-oxidasefrom pumpkin endosperm as a heterologous probe, a GA20-oxidase gene at the GAS locus of Arabidopsis was isolated,encoding a protein that catalyzes the conversion of C20- toC19-GAs. The gaS mutant is probably a null mutant. Expres-sion of GAS is regulated by photoperiod and by apparentend-product repression.

We thank Dr. R. W. Davis for the genomic library, Dr. D. Meinkefor the emb2O mutant, and the Ohio State Arabidopsis BiologicalResource Center for the YACs used in this research. This work wassupported by U.S. Department of Energy Grant DE-FG02-90ER20021 and by U.S. Department of Agriculture Grants 90-37304-6469 and 94-37304-0956 to J.A.D.Z., by Contracts BIOT-CT90-0207and BIOT-CT92-0529 of the BRIDGE programme of the EuropeanUnion to A.J.M.P., and by National Institutes of Health GrantRR-00480 to D.A.G. for the Michigan State University-NationalInstitutes of Health Mass Spectrometry Facility.

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