Ectopic Expression of AINTEGUMENTA inArabidopsis Plants Results in Increased Growth ofFloral OrgansBETH ALLYN KRIZEK*Department of Biological Sciences, University of South Carolina, Columbia, South Carolina

ABSTRACT AINTEGUMENTA (ANT) was previ-ously shown to be involved in floral organ initiation andgrowth in Arabidopsis. ant flowers have fewer andsmaller floral organs and possess ovules that lack integu-ments and a functional embryo sac. The present workshows that young floral meristems of ant plants aresmaller than those in wild type. Failure to initiate the fullnumber of organ primordia in ant flowers may result frominsufficient numbers of meristematic cells. The decreasedsize of ant floral organs appears to be a consequence ofdecreased cell division within organ primordia. Ectopicexpression of ANT under the control of the constitutive35S promoter results in the development of larger floralorgans. The number and shape of these organs is notaltered and the size of vegetative organs is normal.Microscopic and molecular analyses indicate that theincreased size of 35S::ANT sepals is the result ofincreased cell division, whereas the increased sizes of35S::ANT petals, stamens, and carpels are primarilyattributable to increased cell expansion. In addition,35S::ANT ovules often exhibit increased growth of thenucellus and the funiculus. These results suggest that ANTstimulates cell growth in floral organs. Dev. Genet.25:224–236, 1999. r 1999 Wiley-Liss, Inc.

Key words: Arabidopsis flower development; AIN-TEGUMENTA; cell division; cell expansion; AP2/EREBPfamily

INTRODUCTIONFlowers are derived from groups of undifferentiated

cells called floral meristems. Floral organ primordiaarise at defined positions from within these meristems,grow, and eventually differentiate into the four organsof a flower (sepals, petals, stamens, and carpels). Asplant cells do not undergo migration, cell division andcell expansion are the predominant mechanisms bywhich the number and position of organ primordia isdetermined. In addition, the final size and shape of eachorgan is also controlled by these processes. The number,location, and plane of each cell division in the develop-ing organ primordia, as well as the amount and direc-tion of cell expansion, are critically important in deter-

mining the final form of each organ. Although certainaspects of flower development, such as the establish-ment of floral organ identity, are well characterized[reviewed in Coen and Meyerowitz, 1991; Ma, 1994;Sessions et al., 1998; Weigel and Meyerowitz, 1994],very little is known about how the patterns and num-bers of cell divisions are controlled in flowers duringorgan initiation and organ growth [Meyerowitz, 1997].

Furthermore, little is known about the control of cellexpansion during plant development. Cell expansion isknown to be regulated by phytohormones such asauxin, gibberellin, and brassinosteroids [reviewed inCleland, 1987; Hooley, 1996; Metraux, 1987]; mutantsin the biosynthesis or signal transduction pathways ofthese hormones often exhibit defects in cell expansion.Recently, additional genes with roles in controlling cellexpansion have been identified [Hanzawa et al., 1997;Kim et al., 1998; Sablowski and Meyerowitz, 1998;Wilson et al., 1996]. Whereas some genes seem to playgeneral roles in cell expansion in all tissues [Takahashiet al., 1995], others appear to have specific functions inparticular organs [Sablowski and Meyerowitz, 1998] orat particular times in development [Hanzawa et al.,1997], suggesting that cell expansion is controlled bydifferent factors in different tissues.

One gene that is involved in the control of organgrowth during Arabidopsis flower development is AIN-TEGUMENTA (ANT). Mutations in ant result in arandom reduction in floral organ number, the produc-tion of narrow floral organs, and defects in ovuledevelopment including the absence of integuments anda female gametophyte [Baker et al., 1997; Elliott et al.,1996; Klucher et al., 1996; Schneitz et al., 1997]. Thesedefects in both organ initiation and organ growthsuggest that ANT may be involved in regulating cell

Contract grant sponsor: Department of Energy; Contract grant num-ber: 98ER20312.

*Correspondence to: Beth Allyn Krizek, Department of BiologicalSciences, University of South Carolina, Columbia, SC 29208.E-mail: krizek@.sc.edu

Received 3 March 1999; Accepted 1 June 1999

DEVELOPMENTAL GENETICS 25:224–236 (1999)

r 1999 WILEY-LISS, INC.

division in flowers. ANT is a member of the AP2/EREBPfamily of transcription factors, containing two AP2domains of approximately 70 amino acids [Elliott et al.,1996; Klucher et al., 1996]. These domains have beenshown to bind DNA in other members of the AP2/EREBP family [Buttner and Singh, 1997; Kagaya et al.,1999; Liu et al., 1998; Ohme-Takagi and Shinshi, 1995;Stockinger et al., 1997; Zhou et al., 1997]. In addition,ANT has been shown to function as a transcriptionfactor in yeast [Vergani et al., 1997].

ANT is expressed in cotyledon, leaf, floral organ, andovule primordia [Elliott et al., 1996; Klucher et al.,1996]. In flowers, ANT RNAis initially detected through-out organ primordia but later becomes restricted toparticular subdomains within developing organs [El-liott et al., 1996]. In particular, these domains of ANTexpression seem to correlate with regions undergoingactive growth [Elliott et al., 1996]. Ovule primordiaconsist of several morphological regions: the base orstalk of the primordia (funiculus) that connects theovule to the maternal tissue, a central or chalazalregion from which two integuments arise and eventu-ally develop into the seed coat, and the apical tip of theprimordia (the nucellus) in which the megaspore mothercell is produced [reviewed in Reiser and Fischer, 1993].In ovules, ANT is initially expressed throughout theprimordia and later becomes restricted primarily to thechalazal region of the ovule before integument initia-tion [Elliott et al., 1996]. Expression continues in theinteguments during their early development, decreasesas the outer integument grows to cover the nucellus,and eventually becomes limited to the interiormost celllayer of the inner integument [Elliott et al., 1996].

To investigate further the role of ANT in floral organinitiation and growth, ANT was ectopically expressedin wild-type Arabidopsis plants under the constitutivecauliflower mosaic virus 35S promoter (35S::ANT). Themost dramatic phenotype exhibited by 35S::ANT plantsis the production of larger floral organs. The increasedsize of these organs results from an increase in cellnumber in the case of sepals and appears to be largelydue to an increase in cell size in petals, stamens, andcarpels. The relative shape of these organs is main-tained, suggesting that organ size is controlled indepen-dently from organ shape. In addition, 35S::ANT ovulesoften exhibit increased growth of the nucellus andfuniculus but decreased growth of the outer integu-ment. Characterization of ant flowers by two photonfluorescence microscopy shows that ant stage 3 floralmeristems are smaller than wild-type meristems ofsimilar age. Such data provide a possible explanationfor the decreased numbers of organs initiated in antmutants. The smaller size of ant floral organs resultsfrom decreased cell division within floral organ primor-dia. Both the ant mutant and 35S::ANT phenotypes canbe explained by a model in which ANT stimulates cellgrowth.

MATERIALS AND METHODS

Production of 35S::ANT Plants

ANT cDNA was PCR amplified using Pfu polymerase(Stratagene) with primers containing BamHI and XbaIrestriction sites at the 58 and 38 ends of the cDNA,respectively. The polymerase chain reaction (PCR) prod-uct was originally cloned into pLITMUS28 (New En-gland Biolabs), and its sequence was verified by double-stranded sequencing of the recombinant plasmid. ANTwas subsequently subcloned into pGEM3Z containingthe 35S promoter in the KpnI/BamHI sites. 35S::ANTwas cut out of this plasmid with KpnI/XbaI and sub-cloned into the plant transformation vector pCGN1547,which contained a 38 NOS sequence [Krizek and Mey-erowitz, 1996]. 35S::ANT/pCGN-NOS was transformedinto Agrobacterium ASE by electroporation and subse-quently transformed into L-er, ant 26/1, and ant 28/1plants using the in planta vacuum infiltration proce-dure [Bechtold et al., 1993]. Transformants were se-lected by germination of the seeds on MS media contain-ing kanamycin. Putative 35S::ANT ant 26 and 35S::ANT ant 28 plants were genotyped as described laterin Materials and Methods.

Organ Length, Cell Length, and Cell AreaMeasurements

Floral organ lengths were measured using an ocularmicrometer. Two adjacent sepals, two adjacent petals,and two lateral stamens were removed from stage 14flowers (flowers staged as described in Smyth et al.[1990] and Muller [1961]). The length of the carpel wasmeasured after the remaining floral organs were re-moved from each flower. The average size of petal bladecells from L-er and 35S::ANT flowers was determinedusing the software IPLab (Scanalytics, Fairfax, VA).Scanning electron micrograph images were segmentedinto individual cells for quantitation by either handdrawing around the cell or using intensity threshold-ing. The areas of segments corresponding to individualpetal cells were then calculated by IPLab. The averagelength of anther epidermal cells also was determinedusing IPLab. Lines were drawn the middle of each of 13cells from scanning electron micrographs of L-er and35S::ANT anthers. The lengths of these lines were thencalculated by IPLab. For the petal and stamen IPLabmeasurements, four to five SEM image files from four tofive different petals or stamens, respectively, were used.The images used in these measurements correspondedto similar parts of the respective floral organs in theL-er and 35S::ANT flowers.

Scanning Electron Microscopy and Two PhotonFluorescence Microscopy

Samples for SEM were fixed and dried as describedpreviously [Bowman et al., 1991]. For viewing ovules,carpels were sliced with a razor blade immediatelybefore fixation. Flowers and floral organs were mounted

ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH 225

onto stubs, dissected with glass needles as necessary,and coated with gold in a Denton Desk II gold sputter/etch unit. Images were collected on a Hitachi S-2500Dscanning electron microscope and digitally saved usingthe software Iridium (IXRF Systems, Houston, TX).Inflorescence tissue from L-er, 35S::ANT, and ant 25plants was prepared for two photon microscopy asdescribed previously for confocal microscopy [Runninget al., 1995], except that in some cases, the tissue wastreated with RNase at 50 mg/ml for 30 min at 37°C afterfixation and before staining in propidium iodide. Thisstep reduced staining in the cytoplasm. The data werecollected on a Bio-Rad system equipped with a titanium-sapphire laser using a 360 oil immersion lens. Prop-idium iodide was excited at 770 nm, and emitted lightwas collected after passage through filters absorbingwavelengths shorter than 575 nm.

Sequencing and Genotyping of the ant 26 andant 28 Alleles

DNA from ant 26 and ant 28 plants [Baker et al.,1997] was isolated using standard methods. ant wasPCR amplified from each of these DNA samples in fiveoverlapping pieces using either Pfu (Stratagene) orvent (New England Biolabs) polymerase. These PCRproducts were then sequenced directly on a ABI 377automated sequencer. The ant 26 mutation C679=Tresults in a nonsense mutation (Gln227=stop codon).An exon 2 primer was engineered to create an MseI sitespecifically in the ant 26 allele. PCR was performed onleaf tissue [Klimyuk et al., 1993] removed from putative35S::ANT ant 26 plants using this primer and anintron 2 primer. The ant 28 mutation (G1267=A,which results in an Ala =Thr missense mutation)disrupts a PstI site. An intron 7-specific primer andexon 8 primer were used to PCR-amplify this regionfrom candidate 35S::ANT ant 28 plants. Restrictionenzyme analyses distinguished plants that were homo-zygous wild-type, homozygous ant 26 (or ant 28), orheterozygous.

In Situ Hybridization

Flowers for radioactive ANT in situs and nonradioac-tive histone H4 in situs were fixed, embedded, sec-tioned, and hybridized as described previously [Sakai etal., 1995]. The radioactive ANT in situ slides werewashed as described previously [Sakai et al., 1995]. Tomake the ANT antisense probe, a BamHI/ClaI frag-ment of ANT cDNA (corresponding to the 58 half of thegene, not including the AP2 repeats) was cloned into theHincII site of pGEM3Z (pANTsitu). pANTsitu waslinearized by digestion with EcoRI and in vitro tran-scribed in the presence of [35S]UTP with SP6 RNApolymerase. Sections were exposed for 1–2 weeks.Flowers for the nonradioactive histone H4 in situs werewashed as previously described [Coen et al., 1990].Immunological detection of the hybridized probe wasperformed by blocking with 1% Boehringer blocking

agent in phosphate-buffered saline (PBS) with 0.3%Triton X1000 for 1 h, blocking with 0.5% bovine serumalbumin (BSA) in PBS with 0.3% Triton X1000 for 1 h,incubation in anti-DIG antibody diluted 1:1,000 in 0.5%BSA/PBS/0.3% Triton X1000 for 5 h, washing with PBSand subsequently 100 mM Tris pH9.5, 10 mM NaCl, 50mM MgCl2, and color development with NBT/X-phosfor approximately 12 h. Histone H4 antisense probewas made by linearization of pHS-H4 with SpeI and invitro transcription with T7 RNA polymerase in thepresence of digoxigenin-11-UTP (Boehringer-Mann-heim). Histone H4 sense probe was made by lineariza-tion of pHS-H4 with NotI and in vitro transcriptionwith SP6 RNA polymerase in the presence of digoxi-genin-11-UTP. The number of cells expressing histoneH4 in sepals and petals (of stage 7 and 10 flowers) wasdetermined in the following manner. Adjacent tissuesections of appropriately staged flowers that containeda full-length longitudinal section of a sepal or petalwere identified, the number of stained cells in eachtissue section counted, and an average for that indi-vidual organ determined. An overall average was calcu-lated from these individual organ averages.

RESULTS

Ectopic Expression of ANT Increases FloralOrgan Size

After initial expression throughout floral organ pri-mordia, ANT RNA becomes limited to particular subdo-mains as the organs mature [Elliott et al., 1996] (Fig.1A–D). To investigate the consequences of changing thelevels and/or duration of ANT expression in developingorgan primordia, ANT cDNA was fused to the constitu-tive 35S promoter from cauliflower mosaic virus andthis construct was transformed into wild-type Arabidop-sis plants. As shown in Figure 1E–H, 35S::ANT conferspersistent ANT expression in developing floral organs.Approximately one-half of the transgenic plants (66 outof 127 plants) produced larger than normal flowers (Fig.2A). Of the other transformants, most were wild-type inappearance (58), while three had phenotypes resem-bling ant mutants. The plants with ant phenotypes mayarise from cosuppression [reviewed in Depicker andvan Montagu, 1997]. Further characterization wasperformed on those plants which produced the largerflower phenotype. Because these plants are sterile(described later), all phenotypic characterizations weredone on primary transformants. DNA gel blot analysisindicated that some of the 35S::ANT lines with thelarger flower phenotype contained a single copy of thetransgene, whereas others had multiple copies.

The increased size of these 35S::ANT flowers isattributable to increases in the size of all four types offloral organs. Floral organ number is unchanged in35S::ANT plants. The differences in organ size werequantitated by measuring the lengths of floral organsremoved from L-er and 35S::ANT stage 14 flowers

226 KRIZEK

(flowers staged as described in Smyth et al. [1990] andMuller [1961]), using a optical micrometer. These datawere collected from the first 10 flowers on L-er and35S::ANT plants that were grown side by side underequivalent conditions. Results from two different experi-ments are shown in Table 1. Organ size is increasedfrom 11–34% in 35S::ANT flowers. The increased size of35S::ANT floral organs is the opposite phenotype ofthat produced by mutations in ant which cause thedevelopment of smaller floral organs [Elliott et al.,1996; Klucher et al., 1996]. Although organ width wasnot measured, the overall shape of the larger 35S::ANTfloral organs is normal (Fig. 2B). This is not true ofmutations in ant, which tend to produce quite narrowfloral organs [Baker et al., 1997; Elliott et al., 1996;Klucher et al., 1996] (Fig. 2B).

The effects of ectopic ANT expression on organ growthappear to be restricted to floral organs as no differenceswere observed in the size of leaves on 35S::ANT plantscompared with L-er plants or in the height of 35S::ANTplants compared with L-er plants (Fig. 2C). To confirmthat ANT is expressed in these vegetative organs, insitu hybridization was performed on 35S::ANT leaftissue sections. ANT RNA was detected throughout35S::ANT leaf tissue (data not shown).

Other Floral Phenotypes Resulting FromEctopic Expression of ANT

Several other effects on flower development wereobserved in 35S::ANT plants that exhibit the largerfloral organ phenotype. These plants are slightly de-

layed in flowering compared with 35S::ANT plants witha wild-type appearance. Epicuticular wax that is nor-mally present on ovary epidermal cells is not present onthe surface of these cells in 35S::ANT flowers. 35S::ANT plants are male sterile and show severe reduc-tions in female fertility. The anthers do not dehisce;however, this is not the only cause of the male sterilityphenotype. A defect in a late stage of pollen develop-ment appears to occur in 35S::ANT lines. Microsporeswith an exine wall are made and, in at least some cases,viable pollen grains are produced. Pollen viability wasmeasured by staining with the dye fluorescein diacetate(FDA) [Regan and Moffatt, 1990], which assays for theintegrity of the plasma membrane of the vegetative cell[Heslop-Harrison and Heslop-Harrison, 1970]. How-ever, manual cutting of mature 35S::ANT anthers doesnot release individual dehydrated pollen grains asobserved for wild-type anthers. Further work will benecessary to fully characterize pollen development in35S::ANT flowers.

When wild-type pollen is used to fertilize 35S::ANTcarpels, a few seeds are occasionally produced. Toinvestigate the basis for this reduced female fertility,the development of 35S::ANT ovules were character-ized by scanning electron microscopy (SEM). The follow-ing discussion presents a brief description of wild-typeovule development [Robinson-Beers et al., 1992;Schneitz et al., 1995] using the stages assigned inSchneitz et al., 1995. Finger-like ovule primordia areinitiated from the inner ovary walls during stage I ofovule development. During stage II, the inner integu-

Fig. 1. Expression pattern of ANT in L-er and 35S::ANT plants. Insitu hybridization of an ANT antisense RNA probe with longitudinalsections through wild-type (L-er) (A–D) and 35S::ANT tissue (E–H).Each section was photographed in brightfield (A,C,E,G) or darkfield(B,D,F,H). A,B: ANT RNA is detected throughout young floral primor-

dia (arrow). Expression decreases in older flowers. C,D: ANT is nolonger expressed in the sepals or anthers of this stage 10 flower. E,F:ANT RNA is detected throughout the inflorescence of 35S::ANTplants. G,H: ANT RNA is detected throughout this stage 9 35S::ANTflower. se, sepal; pe, petal; st, stamen; ca, carpel.

ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH 227

ment initiates as a symmetrical ring of cells in themiddle or chalazal region of the ovule primordia. Theouter integument initiates soon afterward as an asym-metrical extension of cells on the abaxial surface of theovule just below the inner integument (Fig. 3A,B).During stages II and III, the inner integument expandssymmetrically to cover the nucellus while the outerintegument grows asymmetrically with more growth onthe abaxial surface than the adaxial surface of theovule. The outer integument eventually grows to coverthe inner integument (Fig. 3C) and finally the nucellus(Fig. 3D).

Differences in the development of 35S::ANT ovulesare apparent shortly after the initiation of ovule primor-dia. Although two integuments are initiated in 35S::ANT ovules, they are not as well defined as those inwild-type ovules (Fig. 3E,F). This is particularly truefor the inner integument, which does not protrudesignificantly from the chalazal region of the ovuleprimordia (Fig. 3E). In 35S::ANT ovules, the outerintegument appears to overtake the inner integumentat a slightly earlier time than in wild-type ovules (cf.Fig. 3C,G). Unlike wild-type ovules, in which the outerintegument completely surrounds the nucellus during

Fig. 2. Phenotypes of 35S::ANT, 35S::ANT ant 26, and 35S::ANTant 28 plants. A: Top down and side views of wild-type (L-er) and35S::ANT flowers. B: Petals removed from L-er, 35S::ANT, and ant 29flowers. ant 29 is a strong allele. C: L-er and 35S::ANT plants of equalage. The size of vegetative organs and the overall stature of theseplants are the same. D: ant 26 and 35S::ANT ant 26 flowers showingthe lack of complementation of the organ number and size defects ofant 26. E: Scanning electron micrograph of a typical ant 26 ovule.

There is a slight expansion in the chalazal region of these ovules,where the inner and outer integuments initiate in wild-type ovules. F:Scanning electron micrograph of a 35S::ANT ant 26 ovule showingincreased growth of a single integument-like structure. G: Scanningelectron micrograph of an ant 28 ovule showing partial growth of asingle integumentary-like structure around the nucellus. H: ant 28and 35S::ANT ant 28 flowers showing complementation of the ant 28mutation by 35S::ANT. Scale bars 5 10 µm (E–G).

228 KRIZEK

stage III, the outer integuments of 35S::ANT ovulesrarely grow to surround the nucellus entirely (cf. Fig.3D,H).

A small number (approximately 10%) of mature35S::ANT ovules are wild-type in appearance (cf. Fig. 3Iand J), whereas the rest exhibit growth defects in thedifferent ovule structures. In most 35S::ANT ovules,growth of the outer integument is prematurely termi-nated such that the nucellus is still visible (Fig. 3K).Some of these 35S::ANT ovules resemble ovules of theweak ant 23 allele [Klucher et al., 1996]; however, inmany cases, an unusually large nucellus protrudesfrom the reduced outer integument (Fig. 3L–N). Occa-sionally, ovules with a similar appearance were identi-fied that had a micropyle-like hole visible in the tissueprotruding from the outer integument (Fig. 3Q). Thisfinding suggests that the protruding tissue is the innerintegument; although analysis of the development of35S::ANT ovules indicated that the inner integumentstops growing before the outer integument. As this classof ovule was only observed rarely, it is possible thatsuch a phenotype was missed during examination ofearly ovule development because of the smaller numberof ovules examined. A few 35S::ANT ovules resemblesup ovules [Gaiser et al., 1995] in which there isincreased growth of the outer integument on the ad-axial surface of the ovule (Fig. 3H). In addition to thedefects noted above, the funiculus of 35S::ANT ovules isoften increased in length (Fig. 3N–P). This increasedgrowth appears to primarily result from increased cellelongation (cf. Fig. 3I and P).

35S::ANT Sepals Contain More Cells, Whereas35S::ANT Petals, Stamens, and Carpels Contain

Larger Cells

The increased size of 35S::ANT floral organs couldresult from an increase in cell number, an increase incell size, or some combination of the two. SEM was usedto characterize the size of sepal, petal, stamen, and

carpel epidermal cells in wild-type and 35S::ANT flow-ers. Comparison of cell size in sepals, petals, andcarpels was performed on stage 14 flowers, at whichtime dehiscence of the anthers had already occurred.Stamen cell size was compared in stage 13 flowers inwhich the anthers had just begun to dehisce. Epidermalcells of petals, stamens, and carpels were found to belarger in 35S::ANT flowers compared with L-er flowers(cf. Fig. 4B–E and G–J). Thus, the increased size ofpetals, stamen, and carpels in 35S::ANT flowers is atleast partially attributable to the presence of largercells. No obvious difference in cell size was apparent insepals (Fig. 4A,F), indicating that ectopic expression ofANT results in an increased number of cells in sepals.In general, cell shape and epidermal characteristics areconserved.

To determine whether the increased size of cells inpetals and stamens could account entirely for theincreased size of these organs, scanning electron micro-graphs were analyzed with the graphics software IPLab.The cell area of both the adaxial and abaxial petal bladecells and the length of anther epidermal cells weredetermined (Table 2). The increase in the average cellarea of adaxial and abaxial petal blade cells in 35S::ANT flowers is approximately equal to the increase inpetal size of 35S::ANT flowers, suggesting that in-creased cell expansion can account entirely for theincreased size of these organs. An increase in theaverage length of 35S::ANT anther epidermal cells wassimilar to the overall increase in stamen length, suggest-ing that increased cell expansion may also account forthe increased length of stamens in 35S::ANT flowers,although the relative size of cells in the stamen fila-ment was not determined.

Histone H4 Expression in L-er and35S::ANT Flowers

To investigate further whether the larger size of35S::ANT sepals is due to increased cell division, theexpression pattern of a cell division specific marker wasexamined in L-er and 35S::ANT sepals. Histone H4 haspreviously been shown to be expressed specificallyduring interphase in Antirrhinum floral meristem cells[Fobert et al., 1994] and predominantly during S phaseof the cell cycle in many organisms [Marzluff andPandey, 1988; Nakayama and Iwabuchi, 1993]. Overall,the pattern of histone H4 expression is similar in L-erand 35S::ANT flowers. In both L-er and 35S::ANTinflorescences and flowers, histone H4 is expressed in aspotty pattern, with each spot corresponding to anindividual cell or a small group of cells (Fig. 5A–D). Insepals, petals, and stamens of older flowers (Fig. 5B,D),fewer cells are labeled, indicating decreased numbers ofdividing cells or decreased rates of cell division, or both.These observations are similar to what has been re-ported previously for Antirrhinum [Fobert et al., 1994].No signal was detected with a histone H4 sense probe.

TABLE 1. Floral Organ Lengths (mm) in L-er and35S<ANT Flowers*

Sepals Petals Stamens CarpelsL-er 1.86 6 0.08 3.15 6 0.21 2.51 6 0.16 2.49 6 0.2135S<ANT 2.28 6 0.13 3.95 6 0.27 2.79 6 0.25 3.17 6 0.22% increase 22.7% 25.2% 11.1% 27.6%

L-er 1.88 6 0.14 3.11 6 0.38 2.65 6 0.21 2.54 6 0.3135S<ANT 2.37 6 0.11 4.16 6 0.25 3.02 6 0.29 3.36 6 0.19% increase 25.9% 34.0% 14.1% 32.0%

*The data are indicated as averages 6 SD. The first data set isbased on measurements on two sepals, two petals, two sta-mens, and one carpel from each of 28 L-er flowers on fourplants and each of 45 35S<ANT flowers on 14 plants locatedat positions 1–10 on the inflorescence. The second data set isbased on measurements on two sepals, two petals, two sta-mens, and one carpel from each of 29 L-er flowers on 4 plantsand each of 47 35S<ANT flowers on eight plants located atpositions 1–10 on the inflorescence.

ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH 229

Fig. 3. Scanning electron micrographs of ovule development in L-erand 35S::ANT flowers. Wild-type ovule development (A–D) and amature wild-type ovule (I). 35S::ANT ovule development (E–H) andmature 35S::ANT ovules (J-P). Ovules have been staged according tothe descriptions of Schneitz et al., 1995. A: Wild-type ovules at stage2-IV. The inner integument and outer integument have initiated. B:Wild-type ovules at stage 2-V. Both integuments are growing aroundthe nucellus. C: Stage 3-I wild-type ovules in which the outerintegument is overtaking the inner integument. D: Stage 3-I wild-typeovules in which the outer integuments have enclosed the nucellus. E:35S::ANT ovules in an early stage of development. Both the inner andouter integuments have initiated although they are not as well definedas in wild-type ovules. F: 35S::ANT ovules at stage 2-V, in which theouter integument is beginning to cover the inner integument. G: Stage3-I 35S::ANT ovules in which the outer integument has completely

enclosed the inner integument. H: Stage 3-I 35S::ANT ovules. In somecases, the nucellus continues to protrude from the outer integument. I:Mature wild-type ovule. J: 35S::ANT mature ovule that has a fairlynormal morphology. K: 35S::ANT ovule in which the outer integumenthas not grown to fully enclose the nucellus. L: 35S::ANT ovule inwhich the outer integument does not fully enclose the nucellus and thenucellus has grown abnormally large. M: 35S::ANT ovules with a largenucellus protruding from the outer integument. N: 35S::ANT ovulewith an enlarged nucellus and a long funiculus. O: 35S::ANT ovule inwhich the protruding tissue has an opening resembling a micropyle,suggesting that this is the inner integument. P: 35S::ANT ovule inwhich the outer integument shows increased growth on the adaxialside of the ovule, resembling sup ovules. ii, inner integument; oi, outerintegument; n, nucellus; f, funiculus; m, micropyle. Scale bars 510 µm.

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Fig. 4. Scanning electron micrographs of the epidermal cells on L-er and 35S::ANT floralorgans. Epidermal cells from L-er organs are shown in A–E and corresponding epidermalcells (at the same magnification) from 35S::ANT organs are shown in F–J. A: L-er sepalepidermal cells. B: Epidermal cells on the adaxial surface of a L-er petal. C: Epidermal cellson the abaxial surface of a L-er petal. D: Anther epidermal cells from a L-er stamen. E:

Ovary epidermal cells from a L-er carpel. F: 35S::ANT sepal epidermal cells. G: Epidermalcells on the adaxial surface of a 35S::ANT petal. H: Epidermal cells on the abaxial surface ofa 35S::ANT petal. I: Anther epidermal cells from a 35S::ANT stamen. J: Ovary epidermalcells from a 35S::ANT carpel. Scale bars 5 10 µm.

The number of histone H4-expressing cells in sepalsof L-er and 35S::ANT flowers of stages 6, 7, and 10 wascounted (Table 3). Similar numbers of sepal cells ex-pressing histone H4 were found in L-er and 35S::ANTstage 6 and 7 flowers. However, in sepals from stage 10flowers, more than twice as many 35S::ANT cellsexpressed histone H4 as compared with L-er cells.These results are consistent with the SEM data provid-ing further evidence that increased cell division ac-counts for the increased size of 35S::ANT sepals. Theseresults do not indicate whether the rate of cell divisionin stage 10 35S::ANT sepals is faster than in wild-typesepals or whether the population of dividing cells islarger. For comparison, the number of L-er and 35S::ANT petal cells in stage 10 flowers that expressedhistone H4 was also determined. No significant differ-ences were detected, consistent with earlier resultsindicating that increased cell expansion is primarilyresponsible for the increased size of 35S::ANT petals.

Inflorescence and Floral Meristem Size in L-er,35S::ANT, and ant 25 Plants

To characterize further the effects of ectopic ANTexpression on floral organ growth, two photon fluores-cence microscopy was used to compare the size of youngfloral meristems and young floral organ primordia inL-er and 35S::ANT plants. Stage 3 floral meristems ofL-er and 35S::ANT plants are similar in size (Fig.6A,D), indicating that the increase in organ growth isnot a consequence of a larger floral meristem. Inaddition, the sepal primordia are approximately thesame size in both L-er and 35S::ANT flowers suggestingthat 35S::ANT sepals do not initially consist of morecells. The height and width of stamen and carpelprimordia in stage 6 (Fig. 6B,E) and stage 7 (Fig. 6C,F)L-er and 35S::ANT flowers is also quite similar. Thesedata provide additional evidence that the predominanteffects of ectopic ANT expression on stamens andcarpels is increased cell expansion during later stagesof floral organ development. The effects of ant muta-tions on floral meristem and organ growth were alsoinvestigated by two photon fluorescence microscopy.

Stage 3 ant floral meristems are not as wide as wild-type stage 3 meristems (Fig. 6G). The average width ofant 25 floral meristems was 40 6 4 mm, while that ofwild type was 54 6 1 mm (average 6 standard devia-tion). The carpel primordia in a stage 6 ant 25 flower(Fig. 6H) are approximately the same size as those in aL-er flower. By stage 7, both stamen and carpel primor-dia in ant 25 flowers are thinner than those in wild-type flowers and appear to consist of fewer cell layers(Fig. 6I). These ant 25 organ primordia have grownmore in length than width, changing the shape of theprimordia and making them somewhat difficult tostage. These results are consistent with mutations inant affecting both the numbers and patterns of celldivisions in flowers. SEM analysis of ant 25 maturefloral organs indicates that in almost all cases, the sizeof epidermal cells are the same as in L-er flowers (datanot shown).

Ectopic Expression of ANT in ant 26 and ant 28

Because ectopic expression of ANT sometimes resultsin ovule defects similar to those observed in weak antmutants, the ability of 35S::ANT to complement muta-tions in ant was investigated. Intermediate and weakant alleles (ant 26 and ant 28, respectively) were usedin these studies. ant 26 contains the nucleotide substi-tution C679=T which converts Gln227 into a stopcodon, while ant 28 contains the nucleotide substitu-tion G1267=A, which converts Ala422 into Thr. Inter-estingly, the ant 26 allele displays an intermediatephenotype with slightly more expansion in the chalazalregion of the ovule primordia than found in strong antalleles (Fig. 2E) [Baker et al., 1997], yet contains a stopcodon before the predicted AP2 repeat DNA bindingdomains. The Ala that is mutated in ant 28 is con-served among different members of the AP2 proteinfamily [Okamuro et al., 1997; Riechmann and Meyerow-itz, 1998]. Since 35S::ANT plants are male sterile andmostly female sterile, the 35S::ANT construct wastransformed into ant 26/1 and ant 28/1 plants. Kana-mycin resistant progeny from these transformationswere PCR genotyped [Jacobson and Moscovits, 1991].

Five 35S::ANT ant 26 plants were obtained which allresembled ant 26 plants with regard to floral organnumber and floral organ size (Fig. 2D). However, asingle integumentary-like structure grew around thenucellus in 35S::ANT ant 26 ovules that is not presentin ant 26 ovules (cf. Fig. 2E,F). These 35S::ANT ant 26ovules closely resemble ovules found in the weak ant28 allele (Fig. 2G) [Baker et al., 1997]. Five 35S::ANTant 28 plants were obtained. Three of these plantsresembled 35S::ANT plants with regard to floral organnumber and size (Fig. 2H), while two resembled ant 28plants. The three lines with 35S::ANT-like larger flow-ers also mimicked the other 35S::ANT phenotypes(anther and ovule defects and the absence of epicuticu-lar wax). Thus, ectopic expression of ANT is able topartially rescue the ovule defect of ant 26 plants and

TABLE 2. Average Cell Size (Petals) and Average CellLength (Stamens) in L-er and 35S<ANT Floral Organs*

Petals(adaxial)

Petals(abaxial) Stamens

L-er 1940 6 727 1690 6 471 102 6 2135S<ANT 2458 6 746 2245 6 496 113 6 20% increase 26.7% 32.8% 10.8%

*The data are indicated as averages 6 SD. Measurementunits are pixels. The adaxial petal data are based on measure-ments using 189 L-er and 259 35S<ANT cells from a total offour (L-er) or five petals (35S<ANT). The abaxial petal dataare based on measurements using 132 L-er and 118 35S<ANTcells from a total of four petals each. The stamen data arebased on measurements using 52 L-er and 52 35S<ANT cellsfrom four stamens of each.

232 KRIZEK

rescues both the floral organ and ovule defects of ant 28plants.

DISCUSSION

Ectopic Expression of ANT Resultsin Larger Flowers

Ectopic expression of ANT under a constitutive pro-moter results in increased growth of floral organs. Thisphenotype is the opposite of that resulting from loss ofANT function; ant mutants produce small, narrowfloral organs [Baker et al., 1997; Elliott et al., 1996;Klucher et al., 1996; Schneitz et al., 1997]. The in-creased growth of 35S::ANT floral organs is manifested

as increased cell division in sepals and increased cellexpansion in petals, stamens, and carpels. Owing to thenature of the ANT expression pattern (broad initialexpression with subsequent restriction to particularregions of developing organs), it is unclear whether theincreased growth of 35S::ANT floral organs resultsfrom an increased level of ANT function and/or alengthened period of ANT activity in developing floralorgans. Some evidence for the latter of these possibili-ties is the lack of obvious size differences between L-erand 35S::ANT floral buds of stage 7 and younger.

In ovules, ectopic expression of ANT results in in-creased growth of the proximal (funiculus) and distal(nucellus) elements of an ovule primordia but in de-creased growth of the integuments arising from thechalazal region. In wild-type ovules, an initially broadANT expression domain throughout the primordiumbecomes restricted to the central region of the primor-dium including the chalazal region and the distal partof the funiculus [Elliott et al., 1996]. Thus, the in-creased growth of the nucellus and funiculus in 35S::ANT ovules is likely to result from ectopic expression ofANT in these regions of an ovule. The decreased growthof the outer integument in these ovules may result fromsome sort of integument-specific cosuppression of ANT.Alternatively, growth of the outer integument mayrequires a precise level of ANT expression. Differencesin the levels and patterns of ANT expression in 35S::ANT floral meristems and organs may also explain theinability of 35S::ANT to complement the ant 26 muta-tion.

Fig. 5. Expression of histone H4 in L-er and 35S::ANT flowers. L-er (A,B) and 35S::ANT (C,D). Smallarrows, some of the histone H4 expressing cells in sepals. Wider arrows, petals of stage 10 flowers (B,D).A: Stage 6 L-er flower. B: Stage 10 L-er flower. C: Stage 6 35S::ANT flower. D: Stage 10 35S::ANT flower.se, sepal; pe, petal.

TABLE 3. Number of Cells Expressing Histone H4 RNAin L-er and 35S<ANT Flowers*

Stage 6sepals

Stage 7sepals

Stage 10sepals

Stage 10petals

L-er 7.3 6 2.1 9.6 6 4.1 2.1 6 1.5 21.1 6 6.935S<ANT 8.1 6 2.5 9.1 6 3.1 4.8 6 1.8 22.6 6 3.6

*The data are indicated as averages 6 SD. The stage 6 sepaldata are based on measurements from 12 sepals on 6 L-erflowers and 22 sepals on 11 35S<ANT flowers. The stage 7sepal data are based on measurements from 12 sepals on 6L-er flowers and 12 sepals on 6 35S<ANT flowers. The stage10 sepal data are based on measurements from 17 sepals on 9L-er flowers and 18 sepals on 9 35S<ANT flowers. The petaldata are based on measurements from 11 petals on 11 L-erstage 10 flowers and 13 petals on 9 35S<ANT stage 10flowers.

ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH 233

Fig. 6. Two photon fluorescence microscopy of flower development in L-er, 35S::ANT, andant 25 flowers. L-er (A–C), 35S::ANT (D–F), and ant 25 (G–I). A: L-er stage 3 flower, inwhich the sepal primordia have initiated. B: L-er stage 6 flower. C: L-er stage 7 flower. D:35S::ANT stage 3 flower. E: 35S::ANT stage 6 flower. F: 35S::ANT stage 7 flower. G: ant 25stage 3 flower. The floral meristem is not as wide as a similar stage wild-type floral

meristem. H: ant 25 flower of approximately stage 6. While the carpel primordia aresimilar in size to those of wild type, the stamen primordia appear slightly thinner thanstamen primordia of wild-type flowers. I: ant 25 flower of approximately stage 7. Both thestamen and carpel primordia are thinner those of wild-type; growing more in length thanwidth. Scale bars 5 50 µm.

Comparison of ANT With Other Genes Involvedin Regulating Cell Expansion

The specificity of ANT ectopic expression on alteringcell growth in flowers is similar to ectopic expression ofanother gene, NAP (NAC-like, activated by AP3/PI)[Sablowski and Meyerowitz, 1998]. This gene wasidentified as a target gene of the floral homeotic pro-teins APETALA3 (AP3) and PISTILLATA (PI) and isexpressed in regions of petals and stamens during laterfloral stages (stage 8 to maturity). No NAP mutant hasbeen isolated, but expression of NAP under the 35Spromoter inhibits cell expansion in petals and stamensresulting in shorter organs. Thus NAP and ANT haveopposite effects on cell growth in petals and stamens.NAP has been postulated to function in petals andstamens during the transition from organ growth result-ing from cell division to that resulting from cell expan-sion [Sablowski and Meyerowitz, 1998].

Besides ANT, another member of the AP2/EREBPfamily of transcription factors has been shown to beinvolved in cell growth [Wilson et al., 1996]. The TINYgene seems to play a general role in promoting cellexpansion. tiny plants are short in height, with stemepidermal cells that are reduced in length. tiny flowershave shorter stamens and pistils and rounder buds.Leaf epidermal cells from tiny have a different shapeand are thicker than wild-type cells. Because tiny wasidentified as a dominant mutant in a screen using a Dstransposon containing a 35S promoter, the tiny pheno-type is thought to arise from ectopic expression oroverexpression of the gene [Wilson et al., 1996]. Thus,TINY and ANT appear to have opposite roles in cellgrowth and differ with regard to the organs which areaffected and whether cell shape is altered. tiny, 35S::NAP, and 35S::ANT plants all show reduced fertility[Sablowski and Meyerowitz, 1998; Wilson et al., 1996].

Few mutants or transgenes have been identified thatincrease organ growth. One mutant that causes in-creased growth of leaves and sometimes floral organs isrevoluta (rev) [Talbert et al., 1995]. In the case of leaves,the increase in organ size is due to extra cell divisions,but the basis for the larger floral organs was notreported [Talbert et al., 1995]. Thus, it is unknownwhether rev affects cell expansion in addition to celldivision. The 35S::ANT transgene seems to be currentlyunique in its ability to confer increased organ growththat is specific to flowers. In addition, these plantsexhibit few other phenotypes, none of which occursduring vegetative development. The ability to increasecarpel and ovary size through the use of the 35S::ANTtransgene might prove useful for producing largeryields in agriculture.

Model for ANT Function

ANT is required for floral organ initiation and growth[Baker et al., 1997; Elliott et al., 1996; Klucher et al.,1996; Schneitz et al., 1997]. The results presented in

this paper indicate that ANT is sufficient in flowers fororgan growth but not organ initiation. The decreasednumber of floral organs in ant mutants result from afailure to initiate a normal number of organ primordiaand not from abortion of organ primordia after initia-tion. Since ant floral meristems are smaller than wildtype, the decrease in organ number may result from aninsufficient number of meristematic cells for incorpora-tion into organ primordia. The decreased size of antfloral organs results from changes in the number andorientations of cell divisions within developing floralorgan primordia.

Considering both the loss of function ant mutantphenotype as well as the 35S::ANT ectopic expressionphenotype, the following model describes how ANTmight control organ initiation and growth in flowers.The model proposes that ANT promotes cell growth. Inthe absence of ANT, cell growth is restricted. Studies inyeast and other systems have shown that cells mustreach a critical size before dividing [Mitchison et al.,1997]. Cells below this size threshold are preventedfrom completing the cell cycle. Because of this closerelationship between cell growth and cell proliferation,a decrease in cell growth can cause a reduction in celldivision. The decreased numbers of cells in ant mutantsis thus proposed to be a consequence of decreased cellexpansion. Decreased cell division in ant floral me-ristems and young floral organ primordia ultimatelyleads to the presence of too few cells in young floralmeristems, a reduction in organ initiation, and thedevelopment of smaller floral organs. In 35S::ANTplants, increased ANT function stimulates cell growth.This increased growth results in more cell divisions insepals. In petals, stamens, and carpels increased cellgrowth is not accompanied by increased cell division. Itremians unclear why the effects of increased growth aremanifested in different ways in these different floralorgans. Perhaps other factors involved in carrying outthese growth processes are limiting in different organs.

ACKNOWLEDGMENTSI thank Jannie Lee for doing the histone H4 in situs,

Hajime Sakai for pHS-H4, Charles Gasser for ant 25,ant 26, ant 27, and ant 28 seeds, David Smyth for ant29 seeds and ANT cDNA, Dick Vogt for the use of hiscompound microscope and photography system and forhelp with two photon fluorescence microscopy, MadilynFletcher’s laboratory for the use of IPLab, Dana Dunkel-berger and Shawn Vose for advice on the use of theSEM, and an anonymous reviewer for extremely help-ful comments.

REFERENCESBaker SC, Robinson-Beers K, Villanueva JM, Gaiser JC, Gasser CS.

1997. Interactions among genes regulating ovule development inArabidopsis thaliana. Genetics 145:1109–1124.

ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH 235

Bechtold N, Ellis J, Pelletier G. 1993. In planta Agrobacteriummediated gene transfer by infiltration of adult Arabidopsis thalianaplants. CR Acad Sci 316:1194–1199.

Bowman JL, Smyth DR, Meyerowitz EM. 1991. Genetic interactionsamong floral homeotic genes of Arabidopsis. Development 112:1–20.

Buttner M, Singh KB. 1997. Arabidopsis thaliana ethylene-responsiveelement binding protein (AtEBP), an ethylene-inducible, GCC boxDNA-binding protein interacts with an ocs element binding protein.Proc Natl Acad Sci USA 94:5961–5966.

Cleland RE. 1987. Auxin and cell elongation. In: Davis PJ, editor.Plant hormones and their role in plant growth and development.The Netherlands: Kluwer. p 132–148.

Coen ES, Meyerowitz EM. 1991. The war of the whorls: geneticinteractions controlling flower development. Nature 353:31–37.

Coen ES, Romero JM, Doyle S, Elliott R, Murphy G, Carpenter R.1990. Floricaula: a homeotic gene required for flower developmentin Antirrhinum majus. Cell 63:1311–1322.

Depicker A, van Montagu M. 1997. Post-transcriptional gene silencingin plants. Curr Opin Cell Biol 9:373–382.

Elliott RC, Betzner AS, Huttner E, Oakes MP, Tucker WQJ, GerentesD, Perez P, Smyth DR. 1996. AINTEGUMENTA, an APETALA2-like gene of Arabidopsis with pleiotropic roles in ovule developmentand floral organ growth. Plant Cell 8:155–168.

Fobert PR, Coen ES, Murphy GJP, Doonan J. 1994. Patterns of celldivision revealed by transcriptional regulation of genes during thecell cycle in plants. EMBO J 13:616–624.

Gaiser JC, Robinson-Beers K, Gasser CS. 1995. The ArabidopsisSUPERMAN gene mediates asymmetric growth of the outer integu-ment of ovules. Plant Cell 7:333–345.

Hanzawa Y, Takahashi T, Komeda Y. 1997. ACL5: an Arabidopsis generequired for internodal elongation after flowering. Plant J 12:863–874.

Heslop-Harrison J, Heslop-Harrison Y. 1970. Evaluation of pollenviability by enzymatically induced fluorescence: intracellular hydro-lysis of fluorescein diacetate. Stain Tech 45:115–120.

Hooley R. 1996. Plant steroid hormones emerge from the dark. TrendsGenet 12:281–283.

Jacobson DR, Moscovits T. 1991. Rapid, nonradioactive screening foractivating ras oncogenic mutations using PCR primer-introducedrestriction analysis (PCR-PIRA). PCR Methods Appl 1:146–148.

Kagaya Y, Ohmiya K, Hattori T. 1999. RAV1, a novel DNA-bindingprotein, binds to bipartite recognition sequence through two distinctDNA-binding domains uniquely found in higher plants. NucleicAcids Res 27:470–478.

Kim Q-T, Tsukaya H, Uchimiya H. 1998. The CURLY LEAF genecontrols both division and elongation of cells during the expansion ofthe leaf blade in Arabidopsis thaliana. Planta 206:175–183.

Klimyuk VI, Carroll BJ, Thomas CM, Jones JDG. 1993. Alkalitreatment for rapid preparation of plant material for reliable PCRanalysis. Plant J 3:493–494.

Klucher KM, Chow H, Reiser L, Fischer RL. 1996. The AINTEGU-MENTA gene of Arabidopsis required for ovule and female gameto-phyte development is related to the floral homeotic gene APETALA2.Plant Cell 8:137–153.

Krizek BA, Meyerowitz EM. 1996. Mapping the protein regionsresponsible for the functional specificities of the Arabidopsis MADSdomain organ-identity proteins. Proc Natl Acad Sci USA 93:4063–4070.

Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K,Shinozaki K. 1998. Two transcription factors, DREB1 and DREB2,with an EREBP/AP2 DNA binding domain separate two cellularsignal transduction pathways in drought- and low-temperatureresponsive gene expression, respectively, in Arabidopsis. Plant Cell10:1391–1406.

Ma H. 1994. The unfolding drama of flower development: recentresults from genetic and molecular analyses. Genes Dev 8:745–756.

Marzluff WF, Pandey NB. 1988. Multiple regulatory steps controlhistone mRNA concentrations. Trends Biochem Sci 13:49–52.

Metraux J-P. 1987. Gibberellins and plant cell elongation. In: DavisPJ, editor. Plant hormones and their roles in plant growth anddevelopment. The Netherlands: Kluwer. p 296–317.

Meyerowitz EM. 1997. Genetic control of cell division patterns indeveloping plants. Cell 88:299–308.

Mitchison JM, Novak B, Sveiezer A. 1997. Size control in the cell cycle.Cell Biol Int 21:461–463.

Muller A. 1961. Zur characterisierung der Bluten und Infloreszenzenvon Arabidopsis thaliana (L.) Heynh. Kulturpflanze 9:364–393.

Nakayama T, Iwabuchi M. 1993. Regulation of wheat histone geneexpression. Crit Rev Plant Sci 12:97–110.

Ohme-Takagi M, Shinshi H. 1995. Ethylene-inducible DNA bindingproteins that interact with an ethylene-responsive element. PlantCell 7:173–182.

Okamuro JK, Caster B, Villarroel R, Montagu MV, Jofuku KD. 1997.The AP2 domain of APETALA2 defines a large new family of DNAbinding proteins in Arabidopsis. Proc Natl Acad Sci USA 94:7076–7081.

Regan SM, Moffatt BA. 1990. Cytochemical analysis of pollen develop-ment in wild-type Arabidopsis and a male-sterile mutant. Plant Cell2:877–889.

Reiser L, Fischer RL. 1993. The ovule and the embryo sac. Plant Cell5:1291–1301.

Riechmann JL, Meyerowitz EM. 1998. The AP2/EREBP family ofplant transcription factors. Biol Chem 379:633–646.

Robinson-Beers, Kay P, E. R, Gasser CS. 1992. Ovule development inwild-type Arabidopsis and two female-sterile mutants. Plant Cell4:1237–1249.

Running MP, Clark SE, Meyerowitz EM. 1995. Confocal microscopy ofthe shoot apex. In: Galbraith DW, Bohnert HJ, Bourque DP, editors.Methods in cell biology’’ San Diego: Academic Press. p 217–229.

Sablowski RWM, Meyerowitz EM. 1998. A homolog of NO APICALMERISTEM is an immediate target of the floral homeotic genesAPETALA3/PISTILLATA. Cell 92:93–103.

Sakai H, Medrano LJ, Meyerowitz EM. 1995. Role of SUPERMAN inmaintaining Arabidopsis floral whorl boundaries. Nature 378:199–203.

Schneitz K, Hulskamp M, Kopczak SD, Pruitt RE. 1997. Dissection ofsexual organ ontogenesis: a genetic analysis of ovule development inArabidopsis thaliana. Development 124:1367–1376.

Schneitz K, Hulskamp M, Pruitt RE. 1995. Wild-type ovule develop-ment in Arabidopsis thaliana: a light microscopic study of clearedwhole-mount tissue. Plant J 7:731–749.

Sessions A, Yanofsky MF, Weigel D. 1998. Patterning the floralmeristem. Semin Cell Dev Biol 9:221–226.

Smyth DR, Bowman JL, Meyerowitz EM. 1990. Early flower develop-ment in Arabidopsis. Plant Cell 2:755–767.

Stockinger EJ, Gilmour SJ, Thomashow MF. 1997. Arabidopsisthaliana CBF1 encodes an AP2 domain-containing transcriptionalactivator that binds to the C-repeat/DRE, a cis-acting DNA regula-tory element that stimulates transcription in response to lowtemperature and water deficit. Proc Natl Acad Sci USA 94:1035–1040.

Takahashi T, GaschA, Mishizawa N, Chua N-H. 1995. The DIMINUTOgene of Arabidopsis is involved in regulating cell elongation. GenesDev 9:97–107.

Talbert PB, Adler HT, Parks DW, Comai L. 1995. The REVOLUTAgene is necessary for apical meristem development and for initiatingcell divisions in the leaves and stems of Arabidopsis thaliana.Development 121:2723–2735.

Vergani P, Morandinin P, Soave C. 1997. Complementation of a yeastDpkc1 mutant by the Arabidopsis protein ANT. FEBS Lett 400:243–246.

Weigel D, Meyerowitz E. 1994. The ABCs of floral homeotic genes. Cell78:203–209.

Wilson K, Long D, Swinburne J, Coupland G. 1996. A dissociationinsertion causes a semidominant mutation that increases expres-sion of TINY, an Arabidopsis gene related to APETALA2. Plant Cell8:659–671.

Zhou J, Tang X, Martin GB. 1997. The Pto kinase conferring resistanceto tomato bacterial speck disease interacts with proteins that bind acis-element of pathogenesis-related genes. EMBO J 16:3207–3218.

236 KRIZEK