patterns the Arabidopsis floral meristem and reproductive ... · by Eva Huala (Roe et al., 1997). All other mutant alleles were obtained from the laboratory of Elliot Meyerowitz

4481Development 124, 4481-4491 (1997)Printed in Great Britain © The Company of Biologists Limited 1997DEV0134

ETTIN patterns the Arabidopsis floral meristem and reproductive organs

Allen Sessions1,†, Jennifer L. Nemhauser1, Andy McCall1,‡, Judith L. Roe1, Ken A. Feldmann2

and Patricia C. Zambryski1,*1Department of Plant and Microbiology, 111 Koshland Hall, University of California at Berkeley, Berkeley, CA 94720, USA 2Department of Plant Sciences, University of Arizona, Tucson, AZ 85721, USA †Present address: Department of Biology and Center for Molecular Genetics, University of California at San Diego, La Jolla, CA 92093-0116, USA‡Present address: Department of Biology, Carleton College, Northfield, MN 55057-4000, USA*Author for correspondence

ettin (ett) mutations have pleiotropic effects on Arabidopsisflower development, causing increases in perianth organnumber, decreases in stamen number and antherformation, and apical-basal patterning defects in thegynoecium. The ETTIN gene was cloned and encodes aprotein with homology to DNA binding proteins which bindto auxin response elements. ETT transcript is expressedthroughout stage 1 floral meristems and subsequentlyresolves to a complex pattern within petal, stamen andcarpel primordia. The data suggest that ETT functions toimpart regional identity in floral meristems that affectsperianth organ number spacing, stamen formation, andregional differentiation in stamens and the gynoecium.During stage 5, ETT expression appears in a ring at the topof the floral meristem before morphological appearance ofthe gynoecium, consistent with the proposal that ETT is

involved in prepatterning apical and basal boundaries inthe gynoecium primordium. Double mutant analyses andexpression studies show that although ETT transcriptionalactivation occurs independently of the meristem and organidentity genes LEAFY, APETELA1, APETELA2 andAGAMOUS, the functioning of these genes is necessary forETT activity. Double mutant analyses also demonstratethat ETT functions independently of the ‘b’ class genesAPETELA3 and PISTILLATA. Lastly, double mutantanalyses suggest that ETT control of floral organ numberacts independently of CLAVATA loci and redundantly withPERIANTHIA.

Key words: Arabidopsis, flower development, ETTIN, positionalinformation

SUMMARY

INTRODUCTION

In higher plants, proper flo ral development re q u i res the coor-d i n ated activity of a number of genes that control the pat t e rn-ing of organ type, organ number and organ fo rm (We i ge l ,1995; Ya n o f s ky, 1995). In A rab i d o p s i s, these genes have beencl a s s i fied into those functioning early in the establishment offlo ral meristem identity such as L E A F Y (L F Y), A P E T E L A 1(A P 1), and APETELA2 (A P 2), and those acting later duri n gthe pat t e rning of organ identity such as A P E T E L A 3 (A P 3) ,P I S T I L L ATA (P I) and AG A M O U S (AG) (We i gel andM eye rowitz, 1994). Mutations in these genes affect the flo ra lve rsus inflo resence identity of flo ral shoots and/or the identityof the individual flo ral organs. Most of these genes are thoughtto encode tra n s c ription fa c t o rs (We i gel, 1995; Ya n o f s ky,1995).

There are a number of other mutations which affect organnumber, organ shape and regional differentiation within floralorgans without changes in the identity of either the floral shootor its component organs. For example, mutations in CLAVATA1(CLV1), CLAVATA3 (CLV3), ETTIN (ETT), and PERIANTHIA(PAN) increase organ number within the flower, whereasmutations in TOUSLED (TSL) decrease organ number andorgan size (Clark et al., 1993, 1995, and 1997; Roe et al, 1993

and 1997; Running and Meyerowitz, 1996; Sessions andZambryski, 1995; Sessions, 1997). These genes likely act topattern growth, cell division and regional differentiation duringthe acquisition of identity within the developing flower. CLV1functions partially redundantly with meristem identity genes,whereas PAN and TSL function independently of the meristemand organ identity genes (Clark et al., 1993; Roe et al, 1997;Running and Meyerowitz, 1996).

H e re we describe the isolation of the E T T gene and itsex p ression pat t e rn during early flo ral development. E T Tencodes a protein that is predicted to be nu clear localized andis homologous to DNA binding proteins wh i ch bind to auxinresponse elements (AREs). E T T m R NA is detected in ac o m p l ex pat t e rn throughout early flower development and isconsistent with the proposed role for E T T in pat t e rning: (i)p e rianth organ nu m b e r, and (ii) stamen and carpel fo rm. EarlyE T T ex p ression in the gynoecium suggests that the pro t e i np a rt i c i p ates in the pat t e rning of abaxial tissues and the estab-lishment of apical and basal boundaries in the pri m o rd i u m .I n t e re s t i n g ly, the tra n s c riptional activation of E T T is inde-pendent of known meristem and organ identity functions.L a s t ly, genetic analyses indicate that E T T functions indep e n-d e n t ly of C LV and re d u n d a n t ly with PA N to control flo ra lo rgan nu m b e r.

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MATERIALS AND METHODS

Plant material ett alleles and plant growth conditions were similar to those describby Sessions and Zambryski (1995). ag-5 was isolated and providedby Eva Huala (Roe et al., 1997). All other mutant alleles weobtained from the laboratory of Elliot Meyerowitz (CaliforniaInstitute of Technology).

Scanning electron microscopy (SEM)Fixation, drying and viewing are the same as Sessions (1997).

Identification and isolation of the ETT gene Plasmids, strains and cloning details, as well as data not shownavailable upon request. ETT was isolated using the ett-1 allele. ett-1wasidentified in a large screen of T-DNA mutagenized seed (#75Feldmann, 1991; Azpiroz-Leehan and Feldmann, 1997) and shownSouthern analysis with T-DNA border-specific probes to contain a sinT-DNA insert encoding kanamyacin resistance. The ett mutantphenotype was completely linked to the T-DNA and kanamyacin restance (0 recombinants in 50 homozygous ett-1/ett-1 plants) in F2backcross populations. Left border plasmid rescue of the ett-1 T-DNAborder generated a plant specific 0.4 kb StyI/EcoRI fragment that wasused to screen a Wassilewskija (WsO) lambda genomic library (Roal., 1993). Five clones spanning 25 kb were isolated. Southern anamapped the 0.4 kb StyI/EcoRI border fragment to the center of the 1kb clone ASL2. Clone ASL2 was inserted into transformation vecpSLJ6991 (Jones et al., 1992) as a SacI fragment and introduced directlyinto a suppressed ett-1 line (see below) by vacuum mediated transfomation. ASL2 rescued the ett-1phenotype and subsequently rescued ett-2 and ett-3in crosses, demonstrating that ETTresides within ASL2. Twooverlapping fragments from ASL2, one spanning the predicted insersite in ett-1, were used to screen a Landsberg erecta (LaO) floral andinfloresence specific lambda ZAP cDNA library (Weigel et al., 1992The longest (2.1 kb) of three similar clones, designated number 5, purified as a pBluescript plasmid (pAS13), and sequenced in (GenBank accession no. AF007788). cDNA 5 was used in northanalysis to show the presence of a 2.3 kb transcript in wild-type, ett-2,and ett-3infloresence tips which was absent in ett-1. Sequencing primderived from the cDNA 5 sequence were used to initiate sequencinthe complete 4.5 kb coding region of ETTgenomic DNA from ASL2,corresponding to 300 nucleotides upstream of the predicted transcripstart to 100 bp after the predicted translation stop. Alignment of cDNA 5 sequence with ASL2 sequence suggests that the translationsite lies 77 bp downstream of the 5′ end of cDNA 5. The transcriptionstart site is predicted to lie 341 bp upstream of the predicted translastart site based on RACE and primer extension analysis. The insesight of the left border of the T-DNA in ett-1was determined by sequencing the left border rescue plasmid. 4.0 kb of ett-2, ett-3and ett-4coveringthe complete genomic region was amplified in two fragments by P(Hi Fidelity PCR Kit, Boehringer Mannheim), sequenced in full, anlesions found in fragments cloned from two independent PCR reactlisted in Fig. 2. ett-2was recovered from T-DNA mutagenized WsO see(#1537); ett-3 and ett-4 were isolated from ethylmethane sulfonat(EMS)-treated LaO seed and kindly provided by David Smyth and JoAlvarez (Monash University).

In situ hybridization In situ hybridization using digoxigenin-labeled probes and alkaliphosphatase detection was performed according to the methoDrews (1995) and according to the manufacturer’s (BoehringMannheim) directions. ett-1plants were used as negative controls. ′and 3′ probes were transcribed from pBluescript subclones of pAS(1.6 kb EcoRI (5′), and 0.8 kb BamHI/XbaI (3′).

Generation and identification of double mutants Double mutants were made using the null ett-1allele which is in the

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WsO ecotype. With the exception of lfy-1 and ag-5which are in theColumbia (ColO) ecotype, the remaining mutant alleles (ap1-1, ap2-2, pi-1, ap3-1, ag-1, clv1-1, clv3-1) are in LaO. Control outcrosses ofett-1identified ecotype specific suppressors of ett-1in ColO and LaO,which segregate as single recessive loci in F2 outcross populations:25% of F2 ett-1 plants have a suppressed gynoecium phenotypallowing more valve formation and some seed set. The loci impartsuppression of ett-1 in LaO and ColO have not been mapped. In F2double mutant populations these suppressor loci segregated indedently of ett-1and all other mutations with the exception of lfy-1 andpi-1, which both reside on chromosome 5. Suppressed phenotypedouble mutant populations were distinct and additive, and were scored in the phenotypic analyses presented.

ett-1 was crossed as a male onto the different homozygomutants. Individuals doubly homozygous for ett-1 and the othermutation of interest were identified in five different ways: (i) as new/additive phenotype segregating 1/16 in F2 populations (ap2-2ett-1, ap3-1 ett-1, ett-1 pi-1, ett-1 pan-1); (ii) by Southern analysisof individual F2 mutants to detect ett-1 specific T-DNA inducedRFLPs (ett-1 lfy-1, ag-1 ett-1, ag-5 ett-1); (iii) in F3 populations fromF2 suppressed ett-1 individuals that were heterozygous for the othemutation of interest (ett-1 lfy-1, ag-1 ett-1); (iv) in F3 populationsfrom F2 mutants that were heterozygous for ett-1 (ap1-1 ett-1, clv1-1 ett-1, clv3-1 ett-1, ett-1 pan-1); and (v) by testcrosses (ett-1 pan-1). The ap1-1 enhancing cauliflower-1(cal-1) mutation resident inWsO (Bowman, 1993) was not present in the families segregating ap1-1 ett-1used here.

RESULTS

ett phenotypesWild-type Arabidopsisflowers are composed of four sepalsfour petals, six stamens and a bicarpelate gynoecium (Fig. 1ett mutations only affect the flower, and are pleiotropic: sepand petal number are increased (Fig. 1B), stamen number anther form are decreased (Fig. 1D,E), and the proper diffentiation of gynoecial tissues is significantly altered (Fig. 1GK; Sessions and Zambryski, 1995; Sessions, 1997). ett associ-ated decreases in stamen number are correlated with aberor failed initation of medial stamen primordia (Sessions, 1997whereas the anther defects include a failure in the formationthe interthecal furrow which is often partially formed omissing on the anthers of ett medial stamen (Fig. 1C-E). Theanatomy of tissues and vascular bundles is normal in ett sepals,petals and stamens (not shown).

ett gynoecium phenotypes are allele-strength dependent ainvolve the aberrant development of tissues in place of tovary. Phenotypes for each allele vary. Wild-type gynoecconsist of a stigma-and-style-capped bilocular ovary on an uelongated internode. The ovary is the largest region of the witype gynoecium and is composed abaxially of 2 valves lateraand 2 furrows medially. The phenotypes of ett-2, ett-3and ett-1 gynoecia form a continuum of decreasing ETT function inwhich valve tissue is removed basally (Fig. 1G-K). Lost valvis replaced basally by structures intermediate between abastyle and internode, and medially (between the valves) adaxial style tissue (Fig. 1F-K; Sessions and Zambryski, 199Sessions 1997). These phenotypes have been interpreteresulting from the basalizing of a hypothetical distal abaxiaadaxial boundary, and the raising of a hypothetical proximvalve forming boundary on the gynoecium primordium, in a

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floral organs. (A) Wild-type flower showing 4 petals (p), six stamens (s)he four sepals are not seen in this view. (B) ett-1flower with 5 petals, andns (stamens not visible). (C-K) SEM images. (C) Adaxial surface of a wild-cules separated by the interthecal furrow (arrows); dehiscence occurss. (D,E) Adaxial sides of ett-1anthers showing a reduction in thece of a wild-type gynoecium showing apical stigma (sg), style (st), and laterally, and placentae medially (not shown), atop a reduced internoden the abaxial side of the placenta, between the valves; apical and basal. (G) Gynoecium of a weak ett-2homozygote showing basal reduction innd pronounced outgrowth of the medial ovary in stylar tissues (mo).

strength ett-3homozygote showing a mild ett-3 phenotype of valveonounced medial outgrowths (mo); (in′) internode intermediate between Gynoecium of an intermediate strength ett-3 homozygote expressing are valve reduction (between arrows) and more proliferation (*) of adaxialves; (in′) internode covered primarily in abaxial style tissue. (J) Rarel with a patch of valve tissue (between arrows) bounded apically byells as in K. (K) Typical gynoecium from a strong ett-1homozygote whichy in adaxial style tissue (*) and basally in abaxial style-like tissue (in′).00 µm; H,J, 360 µm.

allele-strength dependent manner (Sessions, 1997). Unlikesepals, petals and stamen, vascular patterning and anatomaltered in ett gynoecia (Sessions and Zambryski, 1995).

Isolation of the ETT geneAn ETT genomic DNA clone was isolated using the T-DNAtagged ett-1 allele, and shown to complement ett-1, ett-2andett-3 (see Materials and Methods). The 2.3 kb ETT transcripthas the potential to encode a 608 amino acid protein witpredicted molecular mass of 66.5 kDa (Fig. 2). The protecontains two serine rich regions and a putative bipartite nuclocalization signal (NLS; Robbins et al., 1991). Furthermothe N-terminal half of ETT shows homology with the DNAbinding domains of the transcription factor ARF1 and threlated protein IAA24 (Ulmasov et al., 1997; Kim et al. 1997These two proteins have been implicated in mediatiresponses at auxin-regulated promoters. Moreover, usingARF1 sequence, a BAC containing the ETT locus was identi-fied in GenBank (U78721); a cDNA isolated with this BACsequence was called ARF3 (Ulmasov et al., 1997). Tsequence of ETTand ARF3are identical with the exception of5 nucleotides. In addition, an 84 amino acid region beginnat position 155 of the ETT protein and containing the NLShighly similar to an ESTfrom rice (D40316, 93%identity). The C-terminalhalf of ETT is unique andof unknown function.

Four ett alleles weresequenced and found tocontain lesions consistentwith the phenotypicseverity of each allele(Fig. 2). The strong ett-1allele has a T-DNAinserted into exon 2 andlacks the wild-type 2.3 kbtranscript as determinedby northern blot andin situ hybridizationanalyses (not shown). Theweak ett-2 allele has asingle bp change thatleads to a conservativeArg to Lys substitution atamino acid 247, and alsoaffects splicing in someett-2 transcripts (J. N. andP. Z., unpublished). Theimproperly spliced tran-script introduces the stopcodon contained in intron5 resulting in a truncatedpolypeptide. The interme-diate strength ett-3 andett-4 alleles containnonsense mutations inexons 8 and 9, respec-tively, downstream of theputative DNA bindingdomain.

Fig. 1. Wild-type and ettflowers and and a bicarpellate gynoecium (g). Ta decrease in the number of stametype anther showing two pairs of lobetween the locules on each thecuintrathecal furrow. (F) Abaxial surfabasal ovary (o) composed of valves(in). Notice the medial furrow (mf) ovalve limits are indicated by arrowsvalve formation (between arrows) a(H) Gynoecium of an intermediate reduction (between arrows) and prtrue internode and abaxial style. (I)strong ett-3phenotype of more sevestyle tissue below and between valgynoecium of a strong ett-1 individuaabaxial style tissue; other abaxial clacks valves and is covered apicallScale bar: C,D,E, 80 µm; E,F,G,I,K, 4

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Expression of ETT in infloresence and floralmeristemsTo determine when and where ETT is expressed during flowerdevelopment, in situ hybridization was performed on floratissues spanning the 13 developmental stages (Smyth et 1990). ETT RNA is first detected before stage 1 in the infloresence meristem (IM) in groups of cells which are developinas new FMs (floral meristems; Fig. 3A-C). The base of thexpression domain tapers and joins the infloresence axis pcambium (Fig. 3A-C). During stage 2, expression resolves procambial tissues in the pedicel, and a domain in the futureceptacle (Fig. 3A-C). This expression appears to mark tsites of vascular differentiation within the floral pedicel anreceptacle (Fig. 3C). In addition, a second patch of expressis detected towards the apex of stage 2 FMs (‘ii’ in Fig. 3C) anthis patch ultimately expands to form a thick ring in the terminmeristem (presumptive gynoecium) during stage 5 (Fig. 3D-FDuring stage 3 and 4, expression in the pedicel remairestricted to the pedicel vasculature in cells that lie between dferentiating phloem and xylem elements (not shown), anappears to be high in presumptive petal and stamen primor(Fig. 3D,E). The expression in the infloresence procambiualso resolves to cells that lie between differentiating phloem a

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1 aaaaggtctaaaagccacaccacacacatcagtcaccagacgtagcagagagcctcactgttgcagagagc 72 actcagtactgttctgtttctctgatacctctctctctcctctctcttttaacattgtccaaattaaaaatctaaactttttttctagtt 162 tttttttttctttaatagaaaagtttttttctccacggcttaaagactcactcatcactgtgctactactctctcttcttttggctgaga 252 gggtaaaagtcatgaagaaactcctctgagttttttttctttctttcttataataaagctcttatctttatctctgtttctctctcctta

M G G L I D L N V M E T E E D E T Q T Q T P S S A S G S V S 30 342 ATGGGTGGTTTAATCGATCTGAACGTGATGGAGACGGAGGAAGACGAAACGCAAACGCAAACACCGTCTTCAGCTTCTGGGTCTGTCTCT

P T S S S S A S V S V V S S N S A G G G V C L E L W H A C A 60 432 CCTACTTCGTCTTCTTCAGCTTCTGTGTCTGTGGTGTCTTCGAATTCTGCTGGTGGAGGGGTTTGTTTGGAGCTGTGGCATGCTTGTGCT

G P L I S L P K R G S L V L Y F P Q G H L E Q A P D F S A A 90 522 GGACCCCTTATCTCTCTACCAAAAAGAGGAAGCCTTGTGTTGTATTTCCCTCAGGGACATTTGGAACAAGCCCCCGATTTCTCCGCCGCG

I Y G L P P H V F C R I L D V K L H A E T T T D E V Y A Q V 120 612 ATTTACGGGCTCCCTCCTCACGTGTTCTGTCGTATTCTCGATGTTAAGCTTCACGCAGAGACGACTACAGATGAAGTTTATGCTCAAGTC

S L L P E S E D I E R K V R E G I I D V D G G E E D Y E V L 150 702 TCTCTTCTTCCTGAGTCAGAGGACATTGAGAGGAAGGTGCGTGAAGGAATTATAGATGTTGATGGTGGAGAGGAAGATTATGAAGTGCTT

K R S N T P H M F C K T L T A S D T S T H G G F S V P R R A 180 792 AAGAGGTCTAATACTCCTCACATGTTTTGCAAAACCCTTACTGCTTCTGATACAAGCACCCATGGTGGTTTCTCTGTTCCTCGCCGAGCT

A E D C F P P L D Y S Q P R P S Q E L L A R D L H G L E W R 210 882 GCTGAGGATTGCTTCCCTCCTCTGGACTATAGCCAGCCCCGGCCTTCTCAGGAGCTTCTTGCTAGGGATCTTCATGGCCTGGAGTGGCGA

F R H I Y R G Q P R R H L L T T G W S A F V N K K K L V S G 240 972 TTTCGCCACATTTATCGAGGGCAACCTAGGAGGCATTTGCTCACTACCGGGTGGAGTGCGTTTGTGAACAAGAAGAAGCTTGTCTCTGGT

D A V L F L R G D D G K L R L G V R R A S Q I E G T A A L S 2701062 GATGCTGTGCTTTTCCTTAGAGGAGATGATGGCAAACTGCGACTGGGAGTTAGAAGAGCTTCTCAAATCGAAGGCACCGCTGCTCTCTCG

A Q Y N Q N M N H N N F S E V A H A I S T H S V F S I S Y N 3001152 GCTCAATATAATCAGAATATGAACCACAACAATTTCTCTGAAGTAGCTCATGCCATATCGACCCATAGCGTTTTCAGCATTTCCTACAAC

P K A S W S N F I I P A P K F L K V V D Y P F C I G M R F K 3301242 CCCAAGGCAAGCTGGTCAAACTTCATAATCCCTGCACCAAAGTTCTTGAAGGTTGTTGACTATCCCTTTTGCATTGGGATGAGATTTAAA

A R V E S E D A S E R R S P G I I S G I S D L D P I R W P G 3601332 GCGAGGGTTGAATCTGAAGATGCATCTGAGAGAAGATCCCCTGGGATTATAAGTGGTATCAGCGACTTGGATCCAATCAGGTGGCCTGGT

S K W R C L L V R W D D I V A N G H Q Q R V S P W E I E P S 3901422 TCAAAATGGAGATGCCTTTTGGTAAGGTGGGACGACATTGTGGCAAATGGGCATCAACAGCGTGTCTCGCCATGGGAGATCGAACCATCT

G S I S N S G S F V T T G P K R S R I G F S S G K P D I P V 4201512 GGTTCCATCTCCAATTCAGGCAGCTTCGTAACAACTGGTCCCAAGAGAAGCAGGATTGGCTTTTCCTCAGGAAAGCCTGATATCCCTGTC

S E G I C A T D F E E S L R F Q R V L Q G Q E I F P G F I N 4501602 TCTGAGGGGATTTGCGCCACAGACTTTGAGGAATCATTGAGATTCCAGAGGGTCTTGCAAGGTCAAGAAATTTTTCCGGGTTTTATCAAC

T C S D G G A G A R R G R F K G T E F G D S Y G F H K V L Q 4801692 ACTTGTTCGGATGGTGGAGCCGGTGCCAGGAGAGGCCGCTTCAAAGGAACAGAATTTGGTGACTCTTATGGTTTCCATAAGGTCTTGCAA

G Q E T V P A Y S I T D H R Q Q H G L S Q R N I W C G P F Q 5101782 GGTCAAGAAACAGTTCCCGCCTACTCAATAACCGATCATCGGCAGCAGCACGGGTTGAGCCAGAGGAACATTTGGTGTGGGCCGTTCCAG

N F S T R I L P P S V S S S P S S V L L T N S N S P N G R L 5401872 AACTTTAGTACACGTATCCTCCCCCCATCTGTATCATCATCACCCTCTTCCGTCTTGCTTACCAACTCGAACAGTCCTAACGGACGTCTG

E D H H G G S G R C R L F G F P L T D E T T A V A S A T A V 5701962 GAAGACCATCACGGAGGTTCAGGTAGATGCAGGCTGTTTGGTTTCCCATTAACCGACGAAACCACAGCAGTTGCATCTGCGACGGCTGTC

P C V E G N S M K G A S A V Q S N H H H S Q G R D I Y A M R 6002052 CCCTGCGTTGAAGGGAATTCCATGAAAGGTGCGTCAGCTGTTCAAAGCAATCATCATCATTCGCAAGGAAGGGACATCTATGCAATGAGA

D M L L D I A L 6082142 GACATGTTGCTAGACATTGCTCTCtagaagggttctttggtttctgtgttttatttgcttgtggcttaagtaaagttcttattttagttg2232 atgatgacttgctgctaacttttggaatgtcacaagttgtgacttatgagagacttgtaaacttggttcaagaatgttctgtgttaggtt2322 caatttaaaaagtgtttgcatcaattccggtt

Fig. 2.Nucleotide and predictedamino acid sequence of ETTtranscript. A composite sequencerepresenting the LaO ETT protein andtranscript derived from genomic andcDNA sequence information. The darkgrey boxes indicate serine-richregions; the light grey box indicates,the putative bipartite nuclearlocalization signa. Closed trianglesrepresent the positions of introns, andthe open triangle represents theposition of the T-DNA insert in exon 2of the ett-1allele. The mutationsfound in the ett-2, ett-3and ett-4alleles are indicated below the wild-type sequence. ett-3and ett-4are fromEMS mutagenized lines and containnonsense mutations from guanine toadenine changes. The domain sharedby ETT with ARE-binding proteinsARF1 and IAA24 includes aminoacids 52 through 391 (Ulmasov et al.,1997; Kim et al., in press). Thelongest cDNA clone begins at nt 265.The first nucleotide represents thepresumed start of transcriptiondeduced from primer extension andRACE analyses. Numbers on left arenucleotides, and amino acids on theright.

xylem elements (Fig. 3L). These results demonstrate that ETTis expressed in a complex pattern throughout early flomeristem formation and floral organ initiation.

Expression of ETT in floral organsETTRNA is not detected in sepal primordia (Figs. 3D-H). ETTtranscript is detected throughout petal primordia during sta4-6, and becomes restricted to procambial cells during sta7 and 8, and ceases by stage 9 (Fig. 3F,K). During stagstamen primordia arise between the receptacle and gynoecrings of expression, and show abaxial expression of ETT tran-script from inception until stage 7 (Fig. 3F,G,H). During stag7-9, ETT transcript is detected in the stamen vasculature ain four bands of cells within each anther: two adaxial stripnear the interthecal furrow and two abaxial stripes betweenlocules and the vasculature (Fig. 3H,J,K). Stamen vascuexpression ceases during stage 9, before morphological difentiation of cell types within the bundles is visible.

Similar to stamen primordia, the gynoecium shows abaxexpression of ETTat inception (Fig. 3F,G). The inner (adaxialedge of the ring of ETT expression in the terminal meristem

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marks the abaxial-adaxial boundary of the gynoecium pmordium before it emerges from the FM (Fig. 3F,G). Expressiofrom stages 5-8 is strictly in the abaxial cells of the gynoeciuprimordium (Fig. 3F-I). This expression is refined during stag9 to cells within the four differentiating vascular strands that lbetween phloem and xylem elements (Fig. 3J,K; not showThis vasculature expression persists until stage 12 (not show

Relationship of ETT to meristem identity functionsIn wild-type plants the IM initiates lateral shoot meristemwhich normally develop as FMs. In meristem identity mutanlateral shoots assume a mixture of floral and infloresenmeristem characters. To determine if any of these genfunctions as an upstream regulator of ETTexpression, doublemutant analyses and expression studies were performed.

LFYLFY is thought to be one of the earliest acting genes in the flomeristem identity program because of loss-of-function phentypes which convert lateral floral shoots toward an infloresenidentity (Huala and Sussex, 1992; Weigel et al., 1992). Usua

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only the late initiated, most apical lateral shoots on stronglfymutants resemble normal flowers, and even these lack peand stamens (Fig. 4A; Huala and Sussex, 1992; Weigel Meyerowitz, 1992). ett-1 lfy-1plants differ from lfy-1 plantsonly in the terminal gynoecia of late initiated lateral shoowhich express the ett phenotype (Fig. 4B). Thus, lfy-1 is largelyepistatic to ett-1except in the most floral like shoots, suggesing that ETTis not active early in the development of lfy-1infloresences and lateral shoots. ETT expression however, islargely normal in a lfy-1 mutant indicating that LFY is notrequired for the early transcriptional activation of ETT in theIM and in lateral meristems (Fig. 4C). Thus some aspect of post-transcriptional function of ETT is dependent on LFY.

AP1AP1 appears to function in concert with LFY in the control ofFM identity (Bowman et al., 1993; Mandel and Yanofsky, 199Weigel and Nilsson, 1995). ap1mutations cause the basal nodeof lateral shoots to have infloresence-like identity, leading to formation of bracts and secondary floral shoots where sepalspetals would normally develop (Fig. 4D). ap1-1 ett-1 lateralshoots have w1 and w2 phenotypes that are identical to thosap1-1,and w3 and w4 phenotypes which are identical to thoof ett-1 (Fig. 4E). ap1-1is epistatic to ett-1in that no more thanfour w1 organs are initiated in ap1-1 ett-1lateral shoots (notshown). Although it sometimes may appear that five w1 grebract-like organs are present in mature ap1-1 and ap1-1 ett-1flowers (Figs. 4D, E), the extra organ develops from the positiof w2 petal primordia (Bowman et al., 1993). ETT thus appearsto not be functioning in w1/w2 of ap1-1 lateral shoots. Similarto the case with lfy-1, ETT expression appears largely normal ap1-1 IMs and FMs (Fig. 4F), suggesting that AP1 is requiredat the posttranscriptional level for ETT activity.

Since LFY, AP1 and CAL, have been shown to be partiallredundant coregulators of meristem identity, expression of ETTwas assayed in ap1-1 lfy-1and ap1-1 cal-1double mutants.ETT expression in lateral meristems occurs in both doubmutants, supporting the conclusion that ETT transcriptionalactivation occurs independently of meristem identity gen(Fig. 4M; not shown).

AP2AP2 is defined as a meristem identity gene due to secondflower production in ap2-1flowers, and the enhancement of lfy

Table 1. Flower organ numbeMutant w1 w2

ap2-2 2.0 (0.0)/1.6 (0.6)1 −ap2-2 ett-1 2.0 (0.2)/1.4 (0.7)1 −ap3-1 4.0 (0.0) 4.0 (0.1)ap3-1 ett-1 4.8 (0.7) 4.4 (0.6)ag-1 4.0 (0.0) 10.1 (0.4)4

ag-1 ett-1 4.1 (4.1) 11.0 (1.4)4

clv3-1 4.5 (0.6) 4.5 (0.6)clv3-1 ett-1 5.7 (0.6) 5.3 (0.7)pan-1 5.6 (0.6) 5.1 (0.7)pan-1 ett-1 4.1 (1.3) 7.6 (1.6)

Standard deviations are given in parentheses.Superscripts indicate organ type as follows: 1medial organs/lateral organs; 2w2

shaped organs; 4w2 and w3 petals not distinguished; 5w4 sepal/petal mosaic or*w3 or w4 organ type as indicated by superscript.

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and ap1 infloresence characteristics by ap2 alleles (Irish andSussex, 1990; Huala and Sussex, 1992; Bowman et al., 19Okamuro et al., 1997). Strong mutations inAP2 such as ap2-2, result in the development of medial w1 carpels in place sepals, the absence of w2 petals, loss of w3 stamens, and osionally unfused w4 carpels (Fig. 4G; Bowman et al., 1991ap2-2 is largely epistatic to ett-1 in w1-w3 of ap2-2 ett-1flowers (Fig. 4H). ett-1does not affect organ number in w1 oap2-2 flowers, but does affect development of w1 carpemargins (Fig. 4I-J). ap2-2 w1 medial carpels generally havecentral valve tissue bounded laterally by a marginal flap medial ovary and stigmatic tissue, and a transmitting tracovered submarginal placenta (Fig. 4I). ap2-2 ett-1w1 medialcarpels lack the marginal flaps, and valve tissue abuts the tramitting tract-covered submarginal placenta (Fig. 4J).

ap2-2 ett-1double mutants suggest that although ETT is notactive in determining organ number in ap2-2, it is required inpatterning differentiation within the margins of w1 ap2-2carpels (Table 1; Fig. 4J). The normal valve development ap2-2 ett-1 w1 medial carpels suggests ETT is not needed forthe development of valve tissue per se, and that its rolenormal w4 gynoecium development is probably to positiowhere valve cell types will form. AP2 does not appear to beinvolved in the early transcriptional activation of ETT, sinceexpression is normal in ap2-2meristems (Fig. 4K). While ETTis not normally expressed in w1 sepals, in ap2-2 ETTRNAappears in the abaxial layers of w1 carpel primordia, suggeing that AP2 is somehow involved in repression of ETTexpression in w1 (Fig. 4L). It is unclear why ETT is transcribedin the abaxial layers of the valve regions of w1 ap2-2carpelswhen it only seems to be functioning in the margins.

Relationship of ETT to organ identity gene functionOrgan identity genes have been typed into a, b and c clas(Bowman et al., 1991). We examined the potential interactiof ETT with members of each class genetically and by in sihybridization.

‘a’ classAP1and AP2, in addition to being classified as meristem identigenes, are also considered ‘a’ class organ identity genes.described above, the organ identity functions of AP1 and AP2appear to act independently of ETT, since ett-1does not affectorgan identity in ap1and ap2mutants (Fig. 4E,H; Table 1).

r in single and double mutantsw3 (w4)* flowers/plants

1.0(0.9)2 45/31.3 (1.1)2 75/5

5.6 (0.6) 0.0 (0.0)3 124/73.8 (1.5) 1.2 (1.2)3 177/10

− 3.8 (0.4)5 52/4− 3.9 (0.5)5 88/6

6.2 (1.1) 0.6 (0.9)6 75/33.4 (2.2) 4.4 (2.1)6 75/35.4 (0.4) 80/83.0 (1.3) 80/8

versus w3 distinctions between stamens was not possible; 3w3 white club-gans; 6w3 staminodes.

4486

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A. Sessions and others

‘b’ class‘b’ class genes are represented by AP3 and PI, mutations inwhich lead to the replacement of petals by sepals and stamby carpels. Flowers from plants homozygous for the weak ap3-1 allele have sepals in w2 and carpeloid stamens in w3 (F5A,B; Bowman et al., 1989). Carpeloid stamens in ap3-1flowers have a variable phenotype but are generally mosaicwhich individual anther locules develop with carpeloifeatures, most noticeably placentae (Bowman et al., 1989; F5B). ap3-1 ett-1flowers have a largely additive phenotype o5 to 6 sepals in each of w1 and w2, w3 organs showing redulocule formation and generally lacking all placental featureand a w4 ett gynoecium (Table 1; Fig. 5C,D). Most notable oap3-1 ett-1flowers is the development in w3 of anther loculelacking club shaped organs covered in anther-like tissue (Ta1; Fig. 5C,D). This phenotype can also be interpreted additive since both ap3-1and ett-1diminish locule formation.

Flowers from plants homozygous for the strong pi-1 allelehave sepals in w1 and w2 and carpeloid organs in w3 and

Fig. 3. In situ hybridization detection of ETTexpression in WsO IMs, FMs, and floral organs. Signalis indicated by blue color. Numbers indicatedevelopmental stage (Smyth et al., 1990). (A) Crosssection through top of IM (I) and young FMs.Expression is detected in clusters of cells that willgrow out as FMs. (B) Serial section 28 µm below thatin A showing the tapered bases of ETT expression thatjoin the procambium of the infloresence axis. Noticepedicel expression in the procambium of the late stage2 FM at top (arrows), and expression throughout stage1 and early stage FMs. Notice early stage 2 FM inlower left showing broader expression at the apicalregions of the FM (receptacle region). Line indicatesplane of section shown in C. (C) Longitudinal sectionthrough the flank of the IM, indicated by a line in B,showing cone-like expression in stage 1 and 2 FMs,and pedicel procambial (arrows) and receptacleexpression in stage 2 FMs. The second patch ofexpression that gives rise to the gynoecium is indicated(ii). (D) Oblique longitudinal section through a stage 4FM showing reticulate expression in traces leading tothe presumptive incipient petals (p) and medialstamens (s), but not the sepals (arrows). Expression inthe second domain (ii) has expanded. (E) Mediallongitudinal section through a stage 4 FM, showingabsence of expression in the sepals (arrows), abaxialexpression in the incipient medial stamen primordia(s), and in the gynoecium primordium (g). (F) Obliquesection through a stage 6 FM showing expression inpetal (p), stamen (s) and the gynoecium (g)primordium. Notice abaxial expression in the stamensand gynoecium. (G) Cross section through a stage 6FM showing abaxial expression of ETT in medialstamen primordia and the gynoecium primordium.Notice absence of expression in sepals. (H) Crosssection through a stage 8 bud showing expression inthe stamens in the procambial strand (v) and in 4patches of cells (arrows) bordering each locule. Expression in thea stage 8 gynoecium showing abaxial expression of ETT transcript. (J) in stamens and the gynoecium except for in the procambial strandshowing expression in procambial strands of petals (p), stamens (axis 300 µm below the top of the IM showing expression in vasculabar: A,B, 35 µm; C, 25 µm; D, 16 µm; E, 18 µm; F, 30 µm; G, 30 µm; H

ens

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w4

(Fig. 5E,F; Hill and Lord, 1989). pi-1w3 organs can be eitherfree from or fused to the central w4 carpels (Fig. 5E,F; Hand Lord, 1989). ett-1 pi-1flowers have an additivephenotype of 5-6 sepals in each of w1 and w2 and ett-likecarpels which lack valve tissues in w3 and w4 (Fig. 5G,HThe additive phenotypes of ett-1 -’b’ class double mutantssuggest that ETT functions independently of ‘b’ functionsduring normal petal and stamen development. Additionalwe have found that ett-1 shows additive interactions withmutations in genes whose wild-type products regulate function activities, including unusual floral organs (ufo)-2,and superman(sup)-1, further suggesting independence oETT and ‘b’ function activity (not shown; Bowman et al.1992; Levin and Meyerowitz, 1995).

‘c’ classAG is the only identified ‘c’ class gene in Arabidopsis. agmutations cause the development of petals and sepals in pof w3 stamens and w4 carpels (Fig. 5I; Bowman et al., 198

gynoecium (g) primordium is abaxial. (I) Medial longitudinal section throughCross section through late stage 8 bud showing reduction in expressions (arrows). (K) Cross section through the base of a bud similar to that in J,ls and ms), and the gynoecium (g). (L) Cross section through the infloresencer strands between differentiating phloem (ph) and xylem (x) elements. Scale, 36 µm; I, 30 µm; J, 36 µm; K, 40 µm; L, 10 µm.

4487ETTIN patterning during flower development

er

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nship of ETT to meristem identity gene function. (A,B,G-J) SEM,K,L,M) In situ hybridization detection of ETTtranscript, signal is blue.nt flower showing carpel-sepal mosaic organs in place of petals and an almost normal gynoecium. (B) ett-1 lfy-1flower showing sepal-carpels surrounding an ettgynoecium (*). (C) ETTtranscript is detected in the

shoot axis and lateral meristems of lfy-1 plants (arrows). (D) ap1-1flowerracts (b) and secondary flowers (sf). (E) ap1-1 ett-1flower similar to thatat it has an ettgynoecium. (F) ETTtranscript is detected in the

shoot axis (i) and lateral meristems (lm) of ap1-1 plants. (G)ap2-2flowerarpels (arrows), an absence of petals, and a reduced number of stamen.1flower showing w1 carpels (arrows) and an ett gynoecium (*). The w1 image are fused to neighboring stamens (common in ap2-2single

Close up of ap2-2w1 carpel margin showing the marginal flap (m) andnal placenta (sm). (J) Close up of ap2-2 ett-1w1 carpel showing the fusedcture (arrow). (K) ETTtranscript is detected in the infloresence shoot axis meristems (lm) of ap2-2 plants, and in the abaxial layers (arrows) of w1p2-2stage 3 FMs (L). (M) ETTtranscript is detected in the lateral p1-1 lfy-1 infloresence shoots. Scale bar: A, 420 µm; B, 450 µm; C, 70 G,H, 1 mm; I, 330 µm; J, 330 µm; K, 45 µm; L, 46 µm; M, 60 µm.

Additionally ag flowers are indeterminate and continue initiaing organs inside the w4 sepals in the whorl-type pattern (pepetals, sepals)n (Fig. 5I). Strong mutations in AG, such as ag-1,are epistatic to ett-1in w3 and w4 (Table 1; Fig. 5J). Weakealleles of ag, such as ag-5, lead to less severe organ identransformations (Roe et al., 1997) and are enhanced by ett-1(Fig. 5K, L). Whereas ag-1 ett-1flowers suggest that ETTis notactive in ag-1, ag-5 ett-1flowers indicate that ETT is necessaryfor full AG activity. In situ hybridization suggeststhat AG plays no role in the transcriptional regu-lation of ETT (Fig. 5M-O). Surprisingly, ETTshows normal abaxial expression in ag-1w3 andw4 organs from inception, as well as abaxialexpression in subsequently initiated organs (Fig.5M-O). The abaxial expression of ETT in ag-1w3-w6 primordia supports the view that ag-1affects organ identity after initiation of normalprimordia (Crone and Lord, 1994). In particular,ag-1w4 primordia which will develop as sepals,express ETT abaxially, similar to carpelprimordia, and not sepal primordia, whichnormally lack ETT expression at inception.

Relationship of ETT function to theorgan number control genes CLV andPAN

CLV CLV1and CLV3are proposed to function togetherto promote differentiation of cells in shootmeristems (Clark et al., 1993, 1995, 1997).Mutations in either gene lead to increases inmeristem size, and to increases in organ numberin all floral whorls (Fig. 6A; Table 1). clv1-1andclv3-1each act additively in double mutant com-bination with ett-1 (Fig. 6B; Table 1; not shownfor clv1-1 ett-1). clv3-1 ett-1flowers had slightincreases in organ number in each whorlcompared to single mutants (Table 1). Stamensand carpels in both double mutants show ett-1-like alterations in regional differentiation of celltypes. Notably, clv3-1 ett-1flowers show a syner-gistic increase in the number of w3 staminodes(reduced stamen), suggesting an interactive rolefor CLV and ETT in the promotion of stamendevelopment (Table 1; Fig. 6B). The indetermi-nacy caused by clv mutations (Clark et al., 1993,1995) which is normally contained within the w4gynoecium is exposed in clv3-1 ett-1flowers dueto the splitting of the style and stigma caused byett-1 (Fig. 6B).

PAN PAN is thought to function to promote bilateralsymmetry within the floral meristem (Runningand Meyerowitz, 1996). pan mutations changeFM symmetry from bilateral to radial, which, likeett mutations, increase sepal and petal number,and decreases stamen number (Fig. 6C). ett andpan mutants differ in gynoecium defects: ettmutants have tissue patterning defects within

Fig. 4. Relatioimages. (C,F(A) lfy-1 mutastamens, andmosaic organinfloresence showing w1 bin D except thinfloresence showing w1 c(H) ap2-2 ett-carpels in thismutants). (I) the submargimarginal stru(i) and lateralprimordia in ameristems ofaµm; F, 50 µm;

t-tals,

rtity

carpels and pan mutants have increases in carpel numb(Running and Meyerowitz, 1996). pan-1 mutant FMs developsimilarly to ett-1 mutants in the increase in adaxial sepal anpetal number and loss of stamen primordia (Fig. 6E,F,H,I,K,Lpan gynoecium development is similar to wild-type with theexception of early trumpet-like as opposed to cylindricgrowth of the gynoecium primordium (Running andMeyerowitz, 1996).

4488

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Fig. 5.Relationship of ETT to organ identity genefunction. (B,D,E-H) SEM images; (M-O) In situhybridization detection of ETT transcript, signal is blue.(A) ap3-1flower showing w1 sepals (se), w2 sepals(se′), and w3 carpeloid stamens (s′). (B) Adaxialsurface of a typical ap3-1w3 carpeloid stamen showingsplit anther (a) and carpel (c) identity. (C) ap3-1 ett-1flower showing w1 and w2 sepals, w3 club shapedorgans (arrow) and an ettgynoecium (*). (D) Abaxialsurfaces of ap3-1 ett-1club-shaped organs and reducedanthers. (E) pi-1 flower with w1 and w2 organsremoved. w3 organs (3) are filamentous and carpeloidand not fused to the central gynoecium. (F) Dissectedpi-1 flower showing the congenital fusion of w3carpeloid organs (3) to the central gynoecium.(G) Dissected ett-1 pi-1flower showing three-lobedcentral gynoecium lacking valves, but covered instigmatic and internode tissue. The organ at left (2) issepaloid and from w2. (H) Dissected ett-1 pi-1flowershowing a three lobed central gynoecium lackingvalves and covered in transmitting tract tissue. Noticew3 organ (3) covered in the same cell-types. (I) ag-1flower showing petals and sepals in place of stamensand carpels, and an indeterminate reiteratingphenotype. (J) ag-1 ett-1flower identical to that in Iexcept bearing 5 organs in each of w1 and w2. (K) ag-5flower showing w3 stamenoid petals and w4 carpeloidsepals. (L) ag-5 ett-1flower showing enhanced agphenotype similar to the strong ag-1allele. (M)Expression of ETTtranscript in a stage 5 ag-1FM issimilar to normal. (N) Expression of ETT transcript instage 6 FMs is also normal. Numbers indicate whorls.(O) Expression of ETT in primordia initiated above w3and w4 also show abaxial expression. Scale bar: B, 180µm; D, 150 µm; E, 400 µm; F, 300 µm; G,H, 360 µm;M,N, 33 µm; O, 45 µm.

ett-1 pan-1 double mutants show synergistic phenotypes, tmost dramatic of which is a loss in the proper spacing andincrease in the number of petal primordia within w2 (Fig. 6DTable 1). Flower development in ett-1 pan -1 double mutantsdiverges from that of ett-1and pan-1 single mutants at stage 3when the sepal primordia initiate (Fig. 6G). Fewer sepprimordia initiate in ett-1 pan-1FMs. These sepals are variablclustered on one side of the FM (Fig. 6G). Aberrant sepinitiation is followed by dramatic proliferation of the remaininFM (Fig. 6G). This proliferation is followed by the initiationof multiple petal primordia, a diminished number of stameprimordia and a trumpet-shaped gynoecium primordium (F6J, M). Development of sepals, stamens and the gynoeciumalso synergistically impaired in ett-1 pan-1double mutants:sepal and stamen primordia often grow into narrow reducorgans, and the gynoecium usually lacks ovules and applike a reduced ett-1gynoecium (not shown).

The enhancement of ett-1 and panphenotypes in doublemutant combination, and the similar w1-w3 phenotypes individual ett and pan mutants suggest that ETT and PAN

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function redundantly in the within-a-whorl spacing of sepaand petal primordia, the initiation of stamen primordia, as weas placental development within the gynoecium. ETT tran-scription in panFMs and IMs is normal (not shown).

DISCUSSION

Based on ettmutant phenotypes and the expression pattern ETT transcripts we propose that ETT has a dynamic role in pat-terning development in groups of cells within floral meristemand reproductive organs. In early patterning, ETT functions indetermining the number of organ primordia, whereas later itinvolved in the outgrowth of and patterning of tissues withiorgan primordia. Mechanistically, how the ETT gene productachieves this is unclear, though it is likely to function as a trascription factor. The homology of ETT with the ARE-bindingproteins ARF1 and IAA24 suggests that ETT may mediaauxin responses at the promoters of auxin regulated ge(Ulmasov et al., 1997; Kim et al., 1997).

4489ETTIN patterning during flower development

ns to

of ETT function to the organ number control genes CLV and PAN. Allages except C and D. (A) Dissected clv3-1flower showing multicarpelateular stamens. (B) clv3-1 ett-1flower showing staminodes (arrows), anderistem (*) emerging through the split style and stigma. (C) pan-1 flowerd stamens. (D) ett-1 pan-1flower showing 10 petals and 3 stamens. The is not visible. (E) pan-1IM (i) and young FMs showing extra abaxialve the normal number of four (arrows). (F) ett-1IM and young FMsat in E. (G) ett-1 pan-1IM and young FMs showing absence of sepalnd proliferation of stage 3 FMs (*). (H) Dissected stage 6 pan-1bud.

emoved to expose the small petal (arrows), stamen (s) and gynoeciumcted stage 6 ett-1bud. 4 of 5 sepals have been removed to expose the, stamen (s) and gynoecium (g) primordia; one medial stamenlar (s′). (J) ett-1 pan-1bud similar in age to H and I showing 2 sepalble the number of petal primordia (arrows), 3 stamen primordia (s) and aium (g). (K) Dissected stage 8 pan-1bud. The sepals have been removedal organ primordia. (L) Dissected stage 8 ett-1bud. Three sepals haveow the inner organ primordia, revealing the small and irregular medial). (M) Undissected ett-1 pan-1bud similar in age to those in K and L,petal primordia, no sepal primordia, and a reduced stamen primordium00 µm; B, 800 µm; E,F, 65 µm; G, 33 µm; H, 60 µm; I, 50µm; J, 60 µm; M, 80 µm.

The pleiotropic nature of ett mutants could result either froma requirement for ETT at an early stage of development whsecondarily affects later stages, or from multiple requiremefor ETT functioning throughout flower developmentExpression patterns and the dissection ofindividual whorl functions in doublemutant combination indicate that ETTfunctions at multiple times during flowerdevelopment and that its activity withineach whorl occurs independently of itsfunctioning in other whorls. For examplew1/w2 and w3/w4 ETT functions can beseparated (ap1-1 ett-1, ag-1 ett-1), andw1/w2/w3 and w4 ETT functions can beseparated (ap2-2 ett-1).

ETT’s role in patterningThe early patterning role of ETT affectsthe number of sepal and petal primordia.ETT expression in the IM occurs beforemorphological appearance of the FM andresolves to a pattern by stage 3 whichmarks the presumptive sites of vasculardevelopment and future organ initiation.Expression in the future pedicel andreceptacle regions of the FM appears lastin cells between differentiating phloemand xylem elements. Defects in theanatomy of ett-1pedicel vascular bundles,however, have not been detected.

The early reticulate expressionsomehow affects the positioning of thesites of sepal and petal initiation withinthe FM, and the sites of their vascularbundles within the pedicel and the recep-tacle, without affecting the size of the FM,or the differentiation of cell types withinvascular bundles, sepals or petals. It iscurious that sepal primordia do notexpress ETT transcript whereas petalprimordia do, since both sepal and petalnumber are increased in ett mutants. Thissuggests that ETTfunctions within thestage 2 FM before sepal and petalprimordia emergence to repress organformation, perhaps by a mechanismrelated to lateral inhibition. ETT appearsto perform this function redundantly withPAN(see below), but independently of theaction of CLV genes, which act moreglobally in shoot meristems to promotethe differentiation of cells. ETT alsoappears to control primordia numberindependently of the TSLprotein kinase(Roe et al., 1997).

The later role of ETT affects theinitiation of and patterning of tissueswithin stamen and carpel primordia.Stamen and carpel primordia express ETTabaxially from inception, and later withinthe developing vascular bundles. Early

Fig. 6.Relationship panels are SEM imgynoecium, and regthe indeterminate mshowing 5 petals anreduced gynoeciumsepal primordia abonearly identical to thprimordia (arrows) aSepals have been rprimordia. (I) Dissesmall petal (arrows)primordium is irreguprimordia, over dougynoecium primordto expose the internbeen removed to shstamen primordia (*showing numerous (*). Scale bar: A, 15µm; K, 70 µm; L, 76

ichnts.

expression of ETT in w3 must function in part to organize indi-vidual primordia, since these primordia often fail to initiate iett mutants (Sessions, 1997). This early expression appearperform a function that requires CLV1and CLV3, and that can

4490

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be compensated for by PAN, to promote the outgrowth ofnormal primordia. CLV1 encodes a putative receptor kinaswhich is absent from stamen primordia but is expressed incenter of stage 4 FMs, perhaps overlapping with ETTexpression (Clark et al., 1997).

Stage 7 stamen primordia additionally express ETT in foursubepidermal vertical streaks in the anther. This antexpression apparently is essential to position where locoutgrowth and the interthecal groove will form as thefunctions are lost in ett-1. The stamen procambial cells expreETT transcript until stage 9, before visible differentiation ovascular cell types, although similar to the petals, stamen vculature appears unaffected in ett mutants. That petal andstamen vasculature is normal in ett mutants suggests that ETTis not functioning in differentiation in these places, or that function is covered by redundant factors in ett mutants.

ETT’s role during gynoecium developmentThe ett allelic series suggests that ETTpatterns the gynoeciumprimordium in a dose-dependent manner. Abaxial expressof ETT in the gynoecium primordium from inception untistage 8 supports its predicted role in prepatterning the prodifferentiation of tissues within the developing organ (Sessioand Zambryski, 1995). The expression data suggest that ETTacts in the abaxial walls of the primordium to perform thressential functions: (i) to promote formation of valve and ovacell types, (ii) to repress formation of stylar and internode ctypes and (iii) to pattern the sites of vascular differentiation aanatomy. One way ETTaccomplishes this is by restricting thactivity of the TSLprotein kinase to the distal gynoecium prmordium (Roe et al., 1997).

We have proposed a model in which the proper differentiatof tissues within the developing gynoecium occurs from twringed boundaries established in the stage 6 gynoecium mordium (Sessions, 1997). Based on this model, ett mutationscause an apical (abaxial-adaxial) boundary to be lowered abasal (valve forming) boundary to be raised on the stage 5/6mordium (Sessions, 1997). This model is in part supportedETTexpression in the gynoecium primordium. For example, inner edge of the ring of ETT-expressing cells in the staggynoecium primordium appears to mark the abaxial-adaxboundary and the hypothetical apical boundary, arguing tETTacts early to directly position this boundary. Similarly, thlower edge of ETTexpression on the stage 5 gynoecium pmordium appears to mark the proposed basal boundary. TETT could act to position these hypothetical developmenboundaries at the edges of it expression domain. AlternativETT could act throughout the gynoecium primordium (i.e. nentirely at the edges) to provide positional information.

ett-1 pi-1flowers suggest that ETT functions similarly in w3carpel primordia. However, the two boundary model is at odwith the phenotype of ap2-2 ett-1w1 carpels, since ETT appearsto be functioning only in the margins of these organs as oppoto throughout the primordium. Perhaps redundant factors present in ap2-2w1 carpel primordia which can promote thformation of valve tissue in the absence of ETT, or there is adifferent developmental basis for carpel development in w1

Independence of meristem and organ identityfunctions from ETT transcriptional activationExpression studies demonstrate that ETT is transcribed inde-

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.

pendently of meristem and organ identity genes. Recent studindicate that LFY and AP1are necessary and sufficient forflower formation within shoot primordia (Mandel and Yanofsky1995; Weigel and Nilsson, 1995), and that AG is necessary andsufficient for the formation of carpels (Mizukami and Ma1992), yet ETTtranscriptional activation occurs in the absencof each of these functions. Additionally, loss of pairs of partialredundant meristem identity functions in ap1-1 lfy-1and ap1-1 cal-1 plants does not appear to alter the early activation ETT transcription in incipient and developing lateral meristemThis argues that other unidentified factors besides the knomeristem and organ identity genes are involved in the trascriptional regulation governing flower development.

While expression studies demonstrate that ETT is expressedin the meristem and organ identity mutants lfy-1, ap1-1, ap2-2 and ag-1, double mutants suggest that ETT is not active inall whorls in these mutant backgrounds. Thus, meristem aorgan identity genes although unnecessary for ETT transcrip-tional activation, are indeed necessary for ETT function. Addi-tionally, ETT appears to be necessary for full AG function,since the partial function of the ag-5 allele requires ETTactivity.

Partial redundancy of ETT and PANSeveral results suggest that ETTand PANfunction redundantlyto control radial patterning within w1-w3. First, the w1-w3phenotype of both single mutants, including the early stage4 floral ontogeny, is very similar. The developmental basis fstamen loss during stage 5 differs between the two mutantsthat ett fails to initiate one of the four medial stamens whilepan mutants initiate 5 equally spaced stamen primord(Running and Meyerowitz, 1996; Sessions, 1997). Second, ettand panmutations have similar genetic interactions in CLV andmeristem and organ identity genes (this study; Running aMeyerowitz, 1996). Third, ett-1 pan-1double mutants showdifferent synergistic phenotypes in each whorl. This is beexemplified in w2 of ett-1 pan-1flowers in the formation ofpetal primordia in all available positions in the whorl. ETTandPANseem to function independently of each other, since eais active in a mutant of the other. Since ett and pan singlemutants do show clear differences, the extent of the redundais unclear. Future experiments expressing ETT in a pan back-ground under constitutive and spatially refined promoteshould help to clarify this relationship.

CONCLUSION

ETT provides a critical function in patterning groups of cellwithin the FM and reproductive organs. Understanding thnature of this patterning function will be aided in the future bgain of function ETTalleles, and experiments which establishwhether ETT acts as a transcription factor and/or mediaauxin-based signals. Transcriptional activation of ETT occursindependently of meristem and organ identity genes, suggeing that other unidentified factors are necessary for flowdevelopment. ETT expression also implies that primordiuminitiation and vascular patterning are coincident events, athat the differentiation of tissues in mature organs is partiapatterned in the meristem before primordia become morphlogically distinct from the meristem.

4491ETTIN patterning during flower development

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We thank John Alvarez and David Smyth for ett-3 and ett-4, EvaHuala for ag-5, Elliot Meyerowitz for clv1-1, clv3-1, and other singlemeristem and organ identity mutants, Ove Nilsson and Detlef Weifor ap1-1 (lfy-6/+), James Keddie for pSLJ6991, the BerkeleElectron Microscope Lab, Steve Ruzin and the NSF center for PlDevelopmental Biology, and Tim Durfee and Fred Hempel for dicussions and critical reading of the manuscript. This work wsupported by Department of Energy grant 88ER13882 to P. C. Z.

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(Accepted 4 September 1997)