Arabidopsisgynoecium structure in the wild type and in … · compare the wild-type pattern of gynoecium cell types and regions, with patterns formed in gynoecia of individuals homozygous

1519Development 121, 1519-1532 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

Arabidopsis gynoecium structure in the wild type and in ettin mutants

R. Allen Sessions* and Patricia C. Zambryski

Department of Plant Biology, 111 Koshland Hall, University of California, Berkeley, Ca, 94720, USA

*Author for correspondence (e-mail: [email protected])

The gynoecium is the female reproductive structure offlowering plants. Here we present a description of the Ara-bidopsis thaliana gynoecium at anthesis. The cylindricalorgan can be broken down into three longitudinal regionsarranged in an apical-basal order: stigma, style, and ovary.Each region can be distinguished histologically and mor-phologically. The transmitting (pollination) tract is axiallypositioned along the center of the gynoecium and spansstigma, style and ovary. Histochemistry, scanning electronmicroscopy and a style-specific reporter gene are used tocompare the wild-type pattern of gynoecium cell types andregions, with patterns formed in gynoecia of individualshomozygous for a series of allelic mutations at the ETTINlocus. ettin gynoecia show morphological and histologicalalterations that appear to result from the merging of apical

and basal regions and the development of abaxial intoadaxial tissues. These developmental abnormalities resultin a reduction of the ovary region, an expansion of thestylar and stigmatic regions, and the abaxial (outward)proliferation of transmitting tract tissue. The alterations inthe mutants can be interpreted as resulting from misspec-ifications of position along the longitudinal and transverseaxes during gynoecium development. The results suggestthat early patterning events underlie wild-type gynoeciumdevelopment, and that ETT functions during this early pro-gramming.

Key words: Arabidopsis, gynoecium, ETTIN, positional information,flower development

SUMMARY

INTRODUCTION

While our understanding of the molecular genetics governingArabidopsis floral meristem and organ identity has increased(recently reviewed by Okamuro et al., 1993; Weigel andMeyerowitz, 1994), regional specification within individualfloral organ types has remained largely unexplored. Manyflowers are composed of concentric whorls of four organ types;the sterile organs of the perianth, the sepals and petals,surround the fertile organs of the androecium and thegynoecium. The reproductive organs exhibit distinct regionaldifferences in shape and composition along their length,making them useful in genetic analyses of development at thesuborgan level.

The gynoecium is the female reproductive organ system andis defined as the collective whorl(s) of carpels, or megasporebearing leaves, that terminate all fruit bearing flowers (reviewedby Gasser and Robinson-Beers, 1993). Along their longitudinalaxis, most gynoecia are composed of three distinct regions: abasal ovary, a style, and an apical stigma. The transmitting (pol-lination) tract and the ovule producing placenta are two addi-tional gynoecium-specific regions. The transmitting tract is acontiguous tissue spanning the entire structure, and is composedof specialized cells that guide the growth of pollen tubes. Withinthe ovary, the pollen tubes deliver sperm to the egg cell-con-taining ovules, which facilitate fertilization and enclose thedeveloping embryos. Most of the cells that make up these fiveregions are functionally and/or structurally distinguishable.

Several mutant loci have been reported that alter gynoeciumdevelopment in Arabidopsis. These include agamous(Bowman et al., 1989), superman (Bowman et al., 1992),unusual floral organs (Wilkinson and Haughn, 1994), fl-82, fl-89, fl-165 (Okada et al., 1989), clavata 1, 2 and 3 (Clark et al.,1994; Alvarez and Smyth, 1994b), ettin (Alvarez, 1994),tousled (Roe et al., 1993), crabs claws (Alvarez, 1994b) andspatula (Alvarez and Smyth, 1994a). The interpretation ofthese mutant phenotypes and of their genetic relationshipshowever, is currently limited by the lack of a cell and tissuelevel description of the wild-type gynoecium.

Here we describe the overall structure of the sexually matureArabidopsis gynoecium excluding the ovules, which havealready been described (Mansfield et al., 1991; Robinson-Beers and Gasser, 1992). We also describe the phenotypesproduced in homozygotes of members of an allelic series ofmutations at the ETTIN (ETT) locus. These results suggest thata spatial prepattern is programmed in the gynoecium pri-mordium and that ETT functions in its establishment and/orelaboration.

MATERIAL AND METHODS

Genetic materials ett-1 and ett-2 were isolated from T-DNA mutagenized seeds of theWassilewskija (WsO) ecotype (Feldmann, 1991). ett-3 was isolatedby John Alvarez and David Smyth from a population of ethylmethane

1520 R. A. Sessions and P. C. Zambryski

Fig. 1. Organization and position of the wild-type gynoecium in the Arabidopsis flower. (A) Stage 15 wild-type flower showing the central postion of the gynoecium (g), its stigma (sg)and the surrounding sepals, petals, and stamen (not labeled). (B) Dehiscing silique (fruit). Seeds(sd), septum (sp), valves (v). (C) Longitudinal section through the lateral plane of an anthesisstage gynoecium stained with PAS and TBO. Ovary (o), ovules (ov), septum (sp), stigma (sg),style (st). (D) Longitudinal section through a stage 12 stigma in the medial plane, stained withPAS and TBO. Elongated stigmatic cells are indicated by arrowheads. (E) Cross sectionthrough an anthesis stage style stained with PAS and TBO. Transmitting tract (t), fans of xylemelements that terminate the medial vascular bundles (sx; better seen in Fig. 4C), chlorenchyma(cl; chloroplasts are the small intracellular dots in the outer 2-3 cell layers subadjacent theepidermis), pollen tubes (pt). (F) Cross section through an anthesis stage ovary stained withPAS and TBO. Lateral (l), medial (m), and intermediate (i) vascular bundles, placenta (pl),ovules (ov), egg sac (e), and septum (sp). The postgenital fusion plane in the septum isindicated by an asterisk. The regions of valve senescence are indicated by arrowheads (only forthe left side of this ovary) and define the limits of a valve. (G) The two carpel model. Scale barin lower left corner: (B) 1 mm, (C) 150 µm, (D) 22 µm, (E) 35 µm, (F) 52 µm.

sulfonate (EMS)-treated Landsberg (LaO)seeds. These three mutations were shown tonot complement in the three pairwise F1 (Fig.5E) and F2 combinations, and to be linked tothe erecta (er) locus on chromosome 2 (notshown). ett-1 has been mapped very near theasymmetric leaves (as) locus on chromosome2 based on the following results: plantshomozygous for er, as and ett-1 were crossedto plants heterozygous for the same mutantloci, in cis. Of the 90 F1 progeny, 48 werewild-type, 35 were er, as, ett-1, 4 were er, and3 were as, ett-1. No recombinants were foundbetween ett-1 and as. ett-1 is not allelic withpinoid mutations, which reside at a locus nearas (not shown). Wild-type plants describedhere are of the WsO ecotype. All plants weregrown in Sunshine Mix soil, in 22°C growthchambers, under 18-hour-light days, andwatered with Miracle Grow plant food.

Thin section histology Tissue was fixed in FAA (50% ethanol, 5%glacial acetic acid, 10% formalin, 1% TritonX-100). Tissue was then dehydrated in ethanoland embedded in JB-4 or JB-4 Plus (Poly-sciences Inc) plastic resin. Sections were cut to3 µm and adhered to glass slides using waterand a slide warmer. Slides were stained in oneof three ways. (i) Periodic-acid-Schiff’s (PAS)reaction followed by toluidine blue (TBO)(Feder and O’Brien, 1968). Slides were airdried and mounted with Permount and a coverslip. Carbohydrates stain a deep pink to red,and other components are stained blue withTBO. (ii) Alcian blue (Pearse, 1980) followedby neutral red (NR). NR staining wasperformed in a 1% aqueous solution for 15seconds. NR was destained with distilledwater. Slides were air dried and mounted withPermount and a cover slip. Acidic polysaccha-rides stain a deep blue. (iii) 0.1% aniline bluein 0.1 M potassium phosphate buffer (pH 7.5or 9), with 40% glycerol and 0.1% n-propylgallate (fluoresence extender) for >30 minutesand mounted in the same solution with a coverslip. Viewed with fluorescence optics (Zeissaxiophot compound light microscope) callosein pollen tubes and phloem elements stains atpH 7.5-9 and appears white and the cuticle ofthe style epidermis stains at pH 9 and appearsorange.

Whole-mount staining Gynoecia were cleared and stained using thesame method to reveal both the vasculature(xylem) and pollen tubes. Gynoecia viewed toassess pollen tube growth were emasculatedbefore anthesis, manually pollinated with WsOpollen, and allowed to grow for 48 hours.Gynoecia were dissected out of flowers, fixedin FAA for 24 hours, washed in 0.1 Mpotassium phosphate buffer (pH 7.5) for 2hours, soaked in 8 M NaOH for 12 hours,rewashed in buffer for 1 hour, and stained in0.1% aniline blue in the same buffer for 30minutes to 2 hours. Viewed with visible light,

1521Arabidopsis gynoecium structure

the xylem in these gynoecia stains a dark blue. Viewed with fluores-cence optics, the pollen tubes in these gynoecia fluoresce bright white.

Scanning electron microscopy (SEM) SEM was performed according to Roe et al., 1993.

GUS assaysThe ASA1:GUS containing line was provided Kris Niyogi and G.Fink. Assays for GUS activity in ASA1:GUS containing plants wereperformed in 50 mM sodium phosphate (pH 7), 0.05% Triton X-100,0.1 mM K3Fe(CN)6, 0.1 mM K4Fe(CN)6, and 1 mM X-glucuronicacid, at 37°C for 12 hours, after an initial 10 minutes vacuum infil-tration of the assay solution. ASA1:GUS containing plants expressGUS at high levels in the style in unemasculated, post anthesisflowers, and at lower levels on the floral peduncle (Niyogi and Fink,in preparation). In our hands style-specific ASA1:GUS reporter geneexpression is only activated in the presence of pollen (i.e. polleninducible). Emasculated flowers from ASA1:GUS plants never stain

Fig. 2. Idioblast position and valve organization. (A) Cleared valvefrom a dehiscent silique, positioned with base at the top of the page,showing vascular patterning of the lateral (l) and intermediate (i)bundles. (B) Longitudinal section through an anthesis stage valve inthe lateral plane stained with PAS and TBO, showing a lateralvascular bundle (l). Labeled cell types from left (outside) to right(inside) include: the outer epidermis (oe), idioblast (id), phloem (ph),xylem (x), those in the layer subadjacent to the inner epidermis(asterisk), and inner epidermis (ie). (C) Longitudinal section throughan anthesis stage valve in the lateral plane stained with PAS andTBO. Intermediate bundle (i) in cross section, chlorenchyma (cl).(D) Cross section through an anthesis stage valve stained with PASand TBO. Lateral vascular bundle (l) and the associated idioblasts(id). (E) Paradermal longitudinal section through the top of ananthesis stage gynoecium in the lateral plane stained with PAS andTBO. Idioblasts (id) develop along the medial vascular bundle (m) atthe junction of the ovary (o) and the style (st). Scale bar in lower leftcorner: (A) 166 µm, (B) 22 µm, (C) 19 µm, (D) 19 µm, (E) 38 µm.

for GUS activity in the style (not shown). Pollen from ASA1:GUSplants does not express GUS activity when grown on wild-type plants(i.e., the GUS expression in the style of ASA1:GUS self pollinatedplants is not from the pollen tubes themselves). Of our GUS assayson ett, ASA1:GUS plants, we performed approximately half on emas-culated flowers that had been pollinated with wild-type pollen, andhalf on self pollinated flowers. Since this reporter is pollen induciblewe tried to cover each gynoecium that we assayed with as much pollenas possible. We also viewed >50 gynoecia of each genotype assayed(>200 total) to see all the possible GUS expressing tissues. Repre-sentative staining patterns are presented in Fig. 11. ASA1:GUS is alsowound inducible to low levels in the vasculature of all floral organs,except the medial vascular bundles of the gynoecium (not shown).This expression however, is extremely low, and not significantrelative to the style tissue expression.

RESULTS

General structure of the wild-type gynoeciumThe Arabidopsis flower is terminated by the gynoecium (Fig.1A). The early development of the Arabidopsis gynoeciuminvolves the formation of a cylinder from the region of the floralmeristem above the medial stamen (Smyth et al., 1990). Laterdevelopment involves the postgenital fusion of the innersurfaces and tip of this cylinder, and the development of ovulesalong the margins of the fused walls, to form a closed bilocularchamber (Hill and Lord 1989; not shown; for an example of asimilar postgenital fusion in the Brassicaceae see Boeke, 1971).After anthesis and fertilization of the egg cells, the gynoeciumelongates approximately sevenfold into the mature seed con-taining silique, or fruit. The silique then dehisces liberating theseeds and the valves (Fig. 1B). The septum is the postgenitallyfused partition and remains on the plant after dehiscence.

StigmaThe stigma is the epidermis of the distal gynoecium and iscomposed of approximately 150, bulbous, elongated epidermalcells that are specialized for pollen attraction and recognition(Fig. 1C, D). The stigma is the only epidermis that will inducepollen germination on an anthesis stage gynoecium(Kandasamy et al., 1994). Following germination, pollen tubesgrow intrusively between the papillar cells into the center ofthe style (Kandasamy et al., 1994). The stigma is the beginningof the transmitting tract.

Style The solid style is a postgenitally fused cylinder composed ofcells specialized for secretion. Polysaccharides are abundant atits distal epidermis (the stigma) and within its axial core asrevealed by staining with PAS (Fig. 1C) and alcian blue (seebelow). PAS and alcian blue stain polysaccharides which arelocalized in the transmitting tract of another member of theBrassicaceae (Hill and Lord, 1987). In Arabidopsis the routesof pollen tubes are tightly correlated with the presence of thePAS and alcian blue staining regions (see below). The stylarcore is composed of axially elongated cells between which thepollen tubes grow (Figs 1E, 3A; Kandasamy et al, 1994), andis the next segment of the transmitting tract after the stigma.This axial core is surrounded by two large xylem arrays,chlorenchyma and a unique stomated epidermis (Fig. 1E; andsee below).


tissue. (A) Medial longitudinal section through pollinated gynoeciumd neutral red (NR). The transmitting tract stains blue, pollen tubes (pt)

ain (pg), stylar xylem (sx). (B) Serial section of A stained with anilined with fluorescent light. Pollen tube (pt) walls and callose plugs (cp)em (sx) is autofluorescent. (C) Alcian blue and NR stained medialgh the septum (sp) in the ovary showing the transmitting tract (t). The the septum is indicated with an asterisk. (D) Cross section through the

blue and NR. Transmitting tract (t), postgenital fusion plane (*), ovulesft corner: (A and B) 38 µm, (C) and (D) 19 µm.

Ovary The ovary is a longitudinally bissected cylinder as seen in crosssection in Fig. 1F and is partitioned by the septum. The axialcenter of the septum is the continuation of the transmittingtract. The apoplasm of these inner septal cells reacts with thePAS (Fig. 1C,F) and alcian blue polysaccharide stains, and thepollen tube detecting callose stain aniline blue (see below). Thepostgenital fusion plane in the septum is denoted by an asteriskand an arrow in Fig. 1F. Alongside the septum are positionedapproximately 45 ovules in four rows. The position of thesenescent zones that will participate in fruit dehiscence (anddefine the limits of a valve) are denoted in Fig. 1F (arrowheads)as are the four main vascular bundles that supply the ovary.

The structure and dehiscence pattern of the Arabidopsisgynoecium is common for many members of the Brassicaceae,whose gynoecia are thought to be composed of two congeni-tally fused carpels that develop in the lateral plane of the flower(reviewed by Okada et al., 1989). According to the two carpelmodel (Fig. 1G), the valves representthe carpels, and the septum, style andstigma represent their postgenitallyfused margins and remaining sub-marginal placentae. With regard tofloral symmetry the gynoecium isoriented in the flower with theseptum in the medial plane (Fig.1F,G). All cross sections in thispaper, unless noted, are oriented withthe medial plane positioned verti-cally.

Gynoecium cell and tissuetypes Certain cells and tissues have proper-ties that distinguish them from othercells and tissues. These distinguish-ing features are useful in comparativeanalyses of positional differences.The cell types described here pertainto their appearance and position atanthesis (stage 13; Smyth et al.,1990).

(a) IdioblastsOne distinguishable cell type is alarge and elongate, phloem associ-ated idioblast found in relativelyinvariant positions in the flower.There are four main vascular bundlesthat supply the gynoecium (Fig. 1F).In the ovary, the idioblasts are onlyassociated with bundles that supplythe valves. A cleared valve from adehiscent silique is shown in Fig. 2Aand stained to reveal the patterning ofthe lateral and intermediate bundles.An anthesis stage gynoecium isshown in longitudinal (Fig. 2B,C)and cross (Fig. 2D) section to revealthe presence of the idioblasts inrelation to the lateral and intermedi-

Fig. 3. Transmitting tract stained with alcian blue anstain a deep red. Pollen grblue, at pH 7.5, and viewefluoresce white. Stylar xyllongitudinal section throupostgenital fusion plane inovary stained with alcian (ov). Scale bar in lower le

ate bundles. The idioblasts are only associated with the medialbundles above the ovary at the junction of the style. Fig. 2Eshows a paradermal section spanning the junction of the styleand ovary demonstrating the distinctive appearance of theseidioblasts in PAS stained sections. The idioblasts in the ovarygenerally stain with a more granular appearance (Fig. 2B) thanthose in the style (Fig. 2E). The idioblasts described here maybe the myrosin cells described by Metcalfe and Chalk (1979)as the only distinctive cell type found in the Brassicaceae.

(b) Valve The simple histology of the valve is also shown in Fig. 2B-D.The valve is six cells thick, unless penetrated by a vascularbundle. In areas lacking a bundle (Fig. 2C,D) there are 4 mor-phologically distinct cell types proceeding from outside in: anouter epidermis, 3 layers of chlorenchyma, a longitudinallyelongated layer subadjacent to the inner epidermis (asterisk),and the radially elongated inner epidermis. The vascular


bundles take the place of the innermost chlorenchyma layer inthe areas in which they develop (Fig. 2B-D).

(c) Transmitting tractThe transmitting tract is distinguishable on the basis of itsalcian blue staining (Fig. 3A,C,D) and its capacity for pollentube growth (as revealed by aniline blue in Fig. 3B). Anilineblue staining reveals the callose in the pollen tube walls, andin the plugs that are laid down proximally as the tip grows (Fig.3B). The transmitting tract cells from style and septum can bedistinguished on the basis of their arrangement. In the septumthey are positioned adjacent to cells arranged in a spongy archi-tecture separated by air spaces (Fig. 3C), whereas in the stylethey are tightly packed and more elongate (Fig. 3A). The trans-

Fig. 4. Scanning electron micrographs and thin section histology ofepidermal tissue types. (A) Top of an anthesis stage gynoeciumshowing the stigma (sg), style(st) and valve (v) surfaces. Pollengrains (pg). (B) The style (st) epidermis is composed of interlockingcells with crenelated wax deposits, and stomata (s). (C) Cross sectionthrough the style stained with aniline blue pH 9. The crenelated waxcuticle exhibits a distinct fluoresence when stained by basic solutionsof aniline blue. The stylar xylem (sx) autofluoresces. (D) Surface ofthe medial side of the ovary. A furrow (large arrow) runs along themedial surface and separates the valves (v), only one of which isshown here. The valves are covered in a mosaic of 4-5 cell clustersthat contain immature unopened stomata (small arrows) at theircenters. (E) Gynoecium base showing the medial surface. Thesurface between the valves (v) is composed of cuboidial cells andstomata. Internode (in). The medial stamen (ms) and the sepal (s)have been removed. The nectary (n) region and the floral pedicel(pd) are indicated. Scale bar in lower left corner: (A) 39 µm, (B) 22µm, (C) 19 µm, (D) 27 µm, (E) 28 µm.

mitting tract is 4-5 cells thick under the inner epidermis (Fig.3C,D), as seen in the septum where the epidermis does not bindalcian blue. The postgenitally fused inner epidermis in the stylehowever, is not obvious, and binds alcian blue (Fig. 3A, seealso 8E).

(d) Epidermal cells The stigma, style and ovary each have a distinct epidermis.Stigmatic cells are characteristically elongate, and bulboustowards the base (Fig. 4A). The epidermis of the style is dis-tinguishable from that of the ovary and stigma by the presenceof open stomates and crenelated wax deposits (Fig. 4B) thatfluoresce when stained by aniline blue in basic pH (Fig. 4C).In the ovary, the valve epidermis is distinguishable from thatof the intervalve region (Fig. 4D). The valve epidermis is apatchwork of 4-5 cell clusters that surround an immature,unopened stomata (Fig. 4D). These stomata open during fruitdevelopment (not shown). The intervalve region is furrowedand covered by small cuboidal cells (Fig. 4D) except at thevery base of the gynoecium, where this surface protrudes outand contains stomata in its epidermis (Fig. 4E).

ettin mutationsThe ETTIN locus is currently defined by ten recessive non-complementing mutant alleles (see Materials and Methods).Structural alterations in the gynoecium are the most apparentphenotypic consequences of the strong ett-1 mutation on floralform (compare Fig. 5A and D) and include: split styles andstigmas, a decrease in ovary size, a reduction in septumformation, tapering of the gynoecium into a stalked likestructure, and outgrowths of tissue from the ovary in the medialplane. Defects in the first three organ whorls also occur inapproximately 50% of ett-1 flowers and include the productionof additional sepals and petals, and some anther loss (notshown). In this paper we focus on the gynoecial abnormalitiesproduced by three representative alleles: ett-1, ett-2, and ett-3(Fig. 5B-D).

Individuals heteroallelic for the three pairwise combinationsof ett-1, ett-2 and ett-3 were constructed to determine relativeallele strength. Using silique length as a measure of ovary size,and hence an estimate of gene function (see below), Fig. 5Edemonstrates that certain alleles will provide some phenotypicrescue in trans to other alleles. The ett-2 allele provides somephenotypic rescue to the ett-1 and ett-3 alleles (comparelengths of ett-2/ett-2, ett-2/ett-3, and ett-3/ett-3 siliques in Fig.5E). Similarly the ett-3 allele provides some phenotypic rescueto ett-1 (Fig. 5E). The ett-1 allele has yet to provide in transphenotypic rescue to any other allele (not shown), and mayrepresent a null mutation. Thus, the order of increasing allelestrength is ett-2<ett-3<ett-1. Fertility differences follow theallelic series: ett-2 homozygotes are fertile, ett-3 homozygotesare slightly fertile, and ett-1 homozygotes are usually sterile.

The differences between wild-type and ett gynoecia can bebroken down into: (i) alterations in vascular patterning, (ii) areduction in ovary size and an outgrowth of transmitting tractin the medial plane, and (iii) the appearance of stylar andstigmatic chacteristics in the ovary region. These alterationswill be considered individually. Additional differencesbetween wild-type and ett gynoecia that will not be consideredin detail here include the elongated internode, or stalk, thatdevelops beneath ett ovaries, and female sterility.


Fig. 5. Scanning electron micrographs of ettin alleles and the relationship between allele strength and valve size. Valve extent is indicated forthe right side of each gynoecium as the space between the two arrows. (A)-(D) Post pollination gynoecia homozygous for ett alleles. (E) Stage18 fruits of homo- and heteroallelic combinations of the ett alleles indicated beneath each fruit. Scale bar in lower left corner: (A) 153 µm, (B)109 µm, (C) 295 µm, (D) 132 µm.

(i) Alterations in vascular patterning in ett gynoeciaWhole-mount staining reveals the four main bundles of thewild-type vascular system (Fig. 6A). The bundles in the lateralplane, which branch to supply the valves (Fig. 2A), terminatejust below the style (Fig. 6A). The medial bundles, which

Fig. 6. Vascular patterning in cleared, whole-mount anthesis stage wild-t(l) vascular bundles are labeled, as is the stylar xylem (sx) that terminatesbifurcation occurs is indicated by an asterisk. The limits of the ovule conarrows. (A) Wild-type gynoecium viewed medially. Lateral bundle termiovary. Only one fan of stylar xylem (sx) is seen in this view. (B) ett-2 gynbundle bifurcation (*), occur below the distal limit of the ovary (the two bMedial bundle bifurcations anastamose with bifurcations of the other medmedially. Medial bundle bifurcation is basalized. Lateral bundles have bi(D) ett-1 gynoecium viewed medially. Notice anastomosed medial bundl(B-D) 166 µm.

supply the placentae, enter the style, and bifurcate into fans ofxylem tissue that are flattened in the lateral plane (Figs 4C,6A). The medial bundles also branch to supply the ovules (Fig.6A). As described above, idioblast association differs betweenmedial and lateral bundles (see Fig. 2).

ype and ett gynoecia. For each gynoecium the medial (m) and lateral the medial bundles. The point along the medial bundle at which the

taining ovary are also indicated in each gynoecium by two largenation and medial bundle bifurcation (*) occur at the distal limit of theoecium viewed laterally. Lateral bundle termination and medialranches of a single medial bundle are not seen in this view). ial bundle in the stylar xylem (sx). (C) ett-3 gynoecium viewed

furcated, and terminate in stylar xylem-like clusters (sx′). Valve (v).e bifurcation branches. Scale bar in lower left corner: (A) 200 µm,


The vasculature of ett gynoecia shows alterations in apical-basal patterning, and a mixing of medial and lateral charactersin the individual bundles. Apical-basal patterning defectsinclude the mispositioning (relative to the distal limit of theovary) of lateral bundle termination and of medial bundlebifurcation (Fig. 6B-D). The mixing of lateral and medialbundle characters occurs in lateral bundles that bifurcate andterminate in medial bundle-like fans of xylem (Fig. 6C).

The severity of the alterations does not parallel the allelicseries. Instead, the vascular patterns produced by the weak andstrong alleles are more similar to each other than to the inter-mediate allele. ett-2 (Fig. 6B) and ett-1 (Fig. 6D) gynoeciashow a similar basalizing of the vasculature with medialbundles splitting and lateral bundles terminating below thedistal limits of the ovary. Unlike the wild type, the bifurcationbranches of each medial bundle in ett gynoecia enter the lateralsides of the ovary and anastomose with the branches of theother medial bundle in the style (Fig. 6B-D). Consistent withallele strength designations, these alterations are more severein ett-1 gynoecia (compare Fig. 6B and D).

The medial and lateral bundles in intermediate strength ett-3 gynoecia are phenotypically variable, and exhibit increasedbranching compared to the wild type. Regarding the basalizedmedial bundle bifurcation, and medial bundle anastamoses, ett-3 gynoecium vasculature patterning is similar to that producedby the weak and strong ett alleles (Fig. 6C). ett-3 medialbundles differ in the variable occurance of branches in theovary region. These branches terminate in style-like arrays thatare found in the distal limits of the ovary (Fig. 6C). The ett-3lateral bundles are phenotypically variable and exhibit bifur-cations, basalized termination and/or termination in a style-type fanned array of xylem. For example, Fig. 6C shows agynoecium in which both lateral bundles have bifurcated andterminated in stylar type arrays that are positioned in themedial outgrowths. The termination of a lateral bundle in astylar-like array is a character normally associated only withmedial bundles.

One consistent alteration in vascular patterning observed inthe three alleles is the association of densely staining idioblastsalong the entire length of the medial bundles (Fig. 7B-D).Idioblasts with this appearance are normally found at the style-ovary junction along the medial bundles (Fig. 2E), and areabsent from the medial bundles in the ovary region (Fig. 7A).As densely staining idioblasts are only associated with themedial bundles at the style-ovary junction in the wild-typegynoecium, the altered association in ett mutants suggests thatett medial tissues in the ovary are more stylar in composition.In summary, the vasculature of ett gynoecia exhibits a generaltrend towards basalization of pattern. This is suggested by thebasalized (relative to the distal limits of the ovary) expressionof lateral bundle termination, medial bundle bifurcation, andidioblast association with the medial bundles.

(ii) Reduction in ovary size and abaxial development oftransmitting tract in ett gynoeciaAs was seen in Fig. 5, as ETT function decreases, the size ofthe ovary decreases. Although the ovary decrease is mostapparent as a reduction of valve tissue (Figs 5, 8A-D), theseptum (Fig. 8C,D,L) and placentae (see below) are alsoreduced. In the apparent place of the missing ovary is seen thedevelopment of everted transmitting tract tissue (Fig. 8C,D,J-L), an internode-like (stalked) structure (Fig. 8B-D) and styletissue (see below).

The everted transmitting tract tissue in ett gynoecia forms inand on outgrowths above (Fig. 8F-H), between (Fig. 8J-K) andbelow (Fig. 8C) sectors of valve tissue, as well as in theapparent place of ‘missing’ valve tissue (Fig. 8D,H and L), inan allele strength-dependent manner. This outwardly facingtransmitting tract is similar to that of the wild type (Fig. 3C)in being 4-5 cells thick under an epidermis (Fig. 8E-L). Thatthese outwardly facing alcian blue-positive tissues can serve asfunctional transmitting tract is demonstrated by pollinating ettgynoecia with wild-type pollen (Fig. 9B-D). Wild-type pollengerminates on the outer epidermis of the outgrowths in ett-2

Fig. 7. Idioblat association with medial bundles in the ovary of ett gynoecia. Cross sections of wild-type and ett gynoecia (through the ovary)stained with PAS and TBO. With increasing allele strength the medial ovary proliferates outward, and the sizes of the valves decrease. Themedial outgrowths (mo) contain idioblasts (id), and transmitting tract (t′) at their periphery (see Fig. 8). (A) Wild-type, showing the absence ofidioblasts along the medial bundle in the ovary. Medial bundle (m), septum (sp), transmitting tract (t), valve (v), xylem (x). (B) ett-2, showingnumerous idioblasts (id) associated with the medial bundle (m) in the medial outgrowth (mo) between the valves. Ovule (ov). (C) ett-3,showing a more pronounced medial outgrowth (unlabeled), branched medial bundles (m′), and numerous idioblasts (id) associated with themedial bundle (m). A pollen grain (p) has landed on the medial outgrowth and germinated. (D) ett-1 showing the absence of valves and thepresence of numerous idioblasts around the entire ovary wall. Arrowheads indicate two ovules. Scale bar in lower left corner: (A) 38 µm, (B)66 µm, (C) 180 µm, (D) 63 µm.



Fig. 9. Pollen tube routes in wild type and ett mutants. Whole-mount gynoecia pollinated with wild-type pollen (p), stained with aniline blueand viewed with fluorescent light. Pollen tubes (pt) grow through tissue that stains positively with alcian blue. (A) Wild-type gynoecium.Pollen (p), pollen tube (pt), stigma (sg), style (st), septum (sp), valve (v). (B) ett-2. Pollen (p) germinates on the medial outgrowth (mo), whichis folded to the side of this gynoecium by a cover slip. Pollen tubes (pt). (C) ett-3. (D) ett-1. Scale bar in lower left: (A-B) 75 µm, (C) 375 µm,(D) 75 µm.

and ett-3 gynoecia (Fig. 9B, C), and along the entire upperovary of ett-1 gynoecia (Fig. 9D). The epidermis of thesetissues cover alcian blue staining subepidermal tissues throughwhich the pollen tubes grow (compare Figs 8 and 9).

The stalked regions of ett gynoecia, although uniform insurface view (Fig. 5B-D), are not uniform internally (Fig. 8B-D). For example, the stalks in ett-1 gynoecia are composed ofa basal region of solid stem (in), a middle region of solid stemformed by a post genital fusion event (in′′), and an apicalregion containing ovules (in′, Fig. 8D). Thus they are not

Fig. 8. Outgrowth of transmitting tract tissue in ett gynoecia.Sections of ett and wild-type gynoecia stained with alcian blue andNR to reveal the transmitting tract (t). Transmitting tract tissues stainblue. With increasing allele strength ett gynoecia undergo thedevelopment of transmitting tract (t′) and a stalk (in, in′, in′′ in placeof valve tissue (delimited by arrowheads to the right of the gynoeciain A-C). Weak alleles result in the outgrowth of this tissue in themedial plane of the gynoecium (mo), whereas intermediate andstrong alleles result in its development in the lateral plane (lo) aswell. (A,E,I) Wild type, (B,F,J) ett-2, (C,G,K) ett-3, (D,H,L) ett-1.(A-D) Longitudinal sections in the lateral plane. (A) Wild-typegynoecium. Transmitting tract (t), internode (in). (B) ett-2gynoecium with expanded internode, and shortened valve. (C) ett-3gynoecium showing reduced valves, lateral outgrowths (lo)containing transmitting tract (t′), and a basal stalk. Although uniformin surface view (see Fig. 5B), this stalk is composed of a lower solidstem (in) and an upper region containing ovules (in′). (D) ett-1gynoecium lacking valves. Transmitting tract tissue (t′) develops intheir place. The lower stalk, although uniform in surface view (seeFig. 5D), is tripartite: a solid basal region (in), a middle solid regionthat has resulted from a postgenital fusion event (in’’), and an upperregion containing ovules (in′). (E-L) Cross sections at levelsindicated in (A-D). With increasing allele strength is seen thedevelopment of transmitting tract tissue (t′) in the medial plane, andits expansion to the lateral sides of the gynoecium. (E-H)Transmitting tract proliferation (t′) in the style is accompanied by abasalized expression of stigmatic tissue (sg′). (E) Wild-type, (F) ett-2, (G) ett-3, (H) ett-1. (I-J) Transmitting tract proliferation in theovary. Medial outgrowth (mo). (I) Wild-type, (J) ett-2, (K) ett-3, (L)ett-1. Scale bar in lower left corner: (A) 180 µm, (B) 280 µm, (C)300 µm, (D) 180 µm, (E)38 µm, (F) 75 µm, (G) 150 µm, (H) 75 µm,(I, J) 75 µm, (K) 188 µm, (L) 38 µm.

entirely true internodes. The complexity of the stalks in otherett gynoecia is allele strength dependent (Fig. 8B,C).

A decrease in ovule production also parallels increases inallele strength. On average (n=10), wild-type WsO and LaOgynoecia have 47 (±4.4) ovules, ett-2 gynoecia have 32 (±3.2)ovules, ett-3 gynoecia have 15 (±3.4) ovules, and ett-1gynoecia have 14 (± 3.9) ovules. The ovules in ett-2 gynoeciaappear morphologically normal and are fully fertile (notshown). Ovules in ett-3 and ett-1 gynoecia are almost com-pletely sterile and are abnormal (not shown). In summary, allaspects of the ovary of ett gynoecia show decreases in size.Unlike the septum and the placentae, the decrease in valveformation can be complete. The appearance of transmittingtract in the outer layers of ett gynoecia suggests that normalabaxial-adaxial polarity has been disrupted, and that outer(abaxial) tissues are developing as if they were inner (adaxial).

(iii) Style and stigma features in the ovary of ettgynoecia

(a) Epidermal charactersThe reduction in the ovary regions of ett gynoecia is coupledwith a basalization of stigmatic and style-like characters. Styleand stigma epidermal cell types are found on the surface out-growths of ett gynoecia. These include style-like cells alongthe flanks of the medial outgrowths of ett-2 (Fig. 10A,B), andett-3 (Fig. 10C, surrounding the valves) gynoecia, and alongthe stalked surfaces of ett-3 and ett-1 gynoecia (Fig. 10D,F,G).Stigmatic cells are found along the medial outgrowths of ett-1and ett-3 gynoecia (Fig. 10C,E). Additionally, an unidentifi-able novel, cell type (asterisk) develops between and beneath(Fig. 10C) the valves in ett-3 gynoecia, and between the apicalstigma and basal stalk in ett-1 gynoecia (Fig. 10E). These arethe cells that cover the alcian blue staining tissues in ettgynoecia and that facilitate wild-type pollen germination. Inett-3 and ett-1 gynoecia they form sharp boundaries with thestyle-like cells on the stalks beneath them (Fig. 10C,F,G).

(b) Style-specific reporter gene expressionAnother character found to exhibit basalized expression in ettgynoecia is the β-glucuronidase (GUS) reporter gene driven bythe Anthranilate Synthetase 1 (ASA1) promoter. In wild-type


Fig. 10. Scanning electron micrographs and thin section histology of epidermal tissue types in ett gynoecia. (A,B) ett-2, (C,D) ett-3, (E-G) ett-1. (A) Surface view of medial outgrowth (mo) of an ett-2 gynoecium in the ovary region, between the valves (v). (B) Cross section of an ett-2gynoecium through the ovary region, stained with aniline blue pH 9, and viewed with fluorescent light. The cuticle of the medial outgrowth(mo) (arrowheads) fluoresces like that of the style. Medial bundle (m), pollen tube (pt), valve (v). (C) Lateral view of an ett-3 gynoecium. Asmall sector of valve (v) is surrounded by a ring of style-like cells (st), and an outer ring of stigmatic (sg) cells that grow along the medialoutgrowth (mo). Below the valve sector are cells of unknown type (asterisk). The epidermis of the stalked region is composed of cells withcrenelated wax deposits (not shown). Open arrow indicates the level of the cross section of D. (D) Cross section of the stalked region of an ett-3gynoecium at the level indicated in Fig. C and stained with aniline blue, pH9. The cuticle (arrowhead) fluoresces like that of the style. Stomate(s). (E) Close up of the medial surface of the upper region of an ett-1 gynoecium. The cells of the medial outgrowth (mo) appear stigmatic (sg).The cells on the lateral sides (asterisks) are of an unknown type, but are similar to the unidentifiable cells in the ett-3 epidermis. (F) Close up ofthe medial surface of an ett-1 gynoecium at the upper limit of the stalked region and the lower limit of the medial outgrowth (mo). The medialoutgrowth cells are smooth and bulbous, whereas those of the stalk have crenelated wax deposits and interlocking shapes like the styleepidermis. There is a noticable boundary around the gynoecium at this level (arrow) between the two cell types. (G) Longitudinal section in themedial plane of an ett-1 gynoecium at the level of the boundary (arrow) indicated in Fig. F, stained with aniline blue and viewed withfluorescent light. Cells below the boundary (arrow) have a cuticle that fluoresces like that of the style epidermis (arrowhead). Scale bar in lowerleft corner: (A) 23 µm, (B) 28 µm, (C) 122 µm, (D) 19 µm, (E) 47 µm, (F) 13 µm, (G) 19 µm.

gynoecia, expression of this reporter gene is confined to theinner layers of the post anthesis stage style (Fig. 11A,B; K.Niyogi and G. Fink, in preparation; see Materials andmethods). ett plants containing the ASA1:GUS sequences showstrong GUS activity in more basal regions of their gynoecia.The tissues showing basalized GUS expression include thoseof the surface outgrowths in ett-2 and ett-3 gynoecia (Fig. 11C-F), and the entire upper ovary wall of ett-1 gynoecia (Fig.11G,H).

Similar to the abaxial development of the normally adaxialtransmitting tract, ASA1:GUS expression is found in the outerlayers of the surface outgrowths of ett gynoecia (Fig.11D,F,H). In contrast to the basalized style epidermis in ettgynoecia, ASA1:GUS expression does not extend along the

entire length of either ett-2 medial outgrowths (Fig. 11C) orthe ett-3 (Fig. 11E) and ett-1 (Fig. 11G) stalked regions. It ispossible that only the epidermal surfaces, and not the internallayers of the lower medial outgrowths of ett-2 gynoecia andthe stalks of ett-3 and ett-1 gynoecia, exhibit the basalizedexpression of style characters.

ettin phenotypic variabilityThe ett phenotypes presented are those found in the highestproportions. There is however some phenotypic variability.This variability is most obvious in the differential expressionof valve tissue along the two lateral sides of the gynoecium. Inall three alleles, each lateral side of the gynoecium has thepotential to develop into a tissue that is different from that of


Fig. 11. ASA1:GUS expression in wild-type and ett gynoecia. (A, C, E, G) Whole-mount gynoecia stained for GUS activity (blue). (B,D,F,H,I)Hand cut sections through stained gynoecia at levels indicated by arrows in (A,C,E,G) showing internal localization of GUS activity. (A,B) Wild-type, (C,D) ett-2, (E,F) ett-3, (G-I) ett-1. Medial outgrowth (mo), ovule (ov), basalized style cells (st′, see Fig. 10C), style xylem(sx), valve (v). Arrow in B indicates region of transmitting tract. Scale bar in lower left: (B,D,F,H,I) 67 µm.

Fig. 12. Phenotypic variability of valve presence and size produced by ett alleles. (A-C) ett-2, (D) ett-3, (E) ett-1. (A) SEM of an ett-2gynoecium in which one lateral side has developed into style-like tissue (st′). Valve (v). (B) SEM of close up of medial surface of gynoecium in A, showing valve (v) side, and style-like (st′) side. A pollen grain has germinated and is seen growing along the style-like surface (arrow).(C) Cross section of a gynoecium similar to that shown in A stained with aniline blue pH 9, and viewed with fluorescent light; the medial planeruns horizontally across the figure. The cuticle of the style-like (st′) side of the gynoecium fluoresces (arrowhead). Pollen tubes (arrows) growin the medial outgrowths (mo) and within the septum. (D) SEM of an ett-3 gynoecium in which no valve tissue has developed on the lateralside. The tissue in its place is style like (st′). Medial outgrowth (mo). (E) SEM of a rare ett-1 gynoecium viewed laterally. A small sector ofvalve (v) bordered by style (st) has developed. Scale bar in lower left: (A) 183 µm, (B) 24 µm, (C) 45 µm, (D) 190 µm, (E) 45 µm.


Table 1. Variability of valve formation in ett gynoecia% Gynoecia with valve tissue on:

allele 2 sides* 1 side neither side

ett-2 (n**=40) 95 5 0ett-3 (n=150) 53 26 21ett-1 (n=40) 0 5 95

*The amount of valve tissue on the two sides of a gynoecium is rarely thesame.

**n= total number of flowers observed from three different plants.

ett-2ETT ett-1ett-3

= trans. tract= style= valve

=medial ovary= internode= idioblast= medial bundle

Fig. 13. Summary of comparative analyses between wild-type and ettgynoecia. Major regions in the wild-type gynoecium and theirrearrangement in gynoecia from plants homozygous for weak (ett-2),intermediate (ett-3), and strong (ett-1) ett alleles. This summaryintegrates information obtained using multiple markers and tissuecharacteristics, and attempts to simplify the data at hand. Eachgenotype is represented by a vertical column of three diagrams,except the wild-type, which has a fourth diagram representing thearrangement of tissues at the style-ovary boundary. Upper rowrepresents surface views and lower rows represent sectional views atlevels indicated by arrows in the upper row. The characteristics ofcell types from five distinct regions of the wild-type gynoecium(leftmost column) are represented by five colors. Color usage asindicated. The transition states between more and less ETT functioncan be seen by proceding from left to right along any row. See text.

the other side. Thus, the two lateral sides of the gynoeciumdevelop independently. This is seen subtly in Fig. 5B and C asa difference in the length of valve tissue that has formed on thetwo lateral sides of ett-2 and ett-3 gynoecia, and also in Fig.11G in an ett-1 gynoecium having a sector of valve tissue thatdoes not express the style-specific GUS reporter gene.

This variability is summarized in Table 1 and in Fig. 12 andis highest for the intermediate strength allele. Whereas ett-2and ett-3 gynoecia usually have valve tissue, ett-1 gynoeciararely do. In ett-2 and ett-3 gynoecia that have valve tissue ononly one lateral side, the other side develops into tissue that isstyle-like (Fig. 12A-D). The nonvalve side has style epidermal(Fig. 12B) and cuticular (Fig. 12C) features. When valve tissuedoes form in ett-1 gynoecia it is always surrounded by a ringof style and stigma epidermis (Fig. 12E) similar to the valvetissue in ett-3 gynoecia (Fig. 10C). The valve-style-stigma jux-taposition of cell types is similar to the wild-type condition,however the arrangement is radial in ett gynoecia whereas it isunidirectional in the wild type.

DISCUSSION

We have described the anthesis stage Arabidopsis gynoecium.This structure is composed of at least 5 regions of distin-guishable tissues: the stigma, style, valve, placentae and trans-mitting tract. We have used this description to analyze the phe-notypes produced by a series of mutations at the ETTIN locuswhich result in alterations in the normal patterning of tissuesin the developing gynoecium (Alvarez, 1994).

The comparison of ett and wild-type gynoecia shows that areduction in the ovary and a basalization of stigma- and style-like tissues occurs as ETT function decreases. This suggeststhat ETT is involved in apical-basal gynoecium patterning thatensures the proper formation of the ovary and the restrictionof stigma and style cell differentiation to the apical end of thedeveloping gynoecium. Further, the abaxial development ofthe normally adaxially positioned transmitting tract in ettgynoecia suggests that ETT is also involved in the establish-ment of normal adaxial-abaxial polarity. The range of allelestrengths and their resultant phenotypes suggest that ETTperforms these functions in a dose dependent manner, ratherthan by a simple threshold effect mechanism.

A color coded summary presented in Fig. 13 comparesgynoecial organization between the wild type and ettin mutantsand attempts to account for surface (top row) and internal(bottom rows) morphological and anatomical alterations thatoccur in gynoecia with decreasing amounts of ETT function(from left to right). In general, the ovary loss accompanyingthe decrease of ETT function is associated with progressive

basalization of those characters represented by yellow and bluein Fig. 13 in the apical-basal dimension, and an increasedinversion of those internal characters represented by blue in theadaxial (inside)-abaxial (outside) dimension. The ovaryreduction is easiest seen in Fig.13 as a decrease in the devel-opment of valve tissue (pink areas) as ETT function is lost,although reduction in the septum and placenta also occurs.

Basalization of apical gynoecium characters in ettgynoeciaBasalization is suggested by the appearance of stigmatic andstylar characteristics in the ovary regions of ettin gynoecia.These characteristics (indicated as blue papillae and yellowcolor in Fig. 13) include those of the epidermis, the vascula-ture, and style-specific reporter gene expression. Althougheach ett allele produces a different pattern of basalized features,the mispatterning of more apical features follows a general rule


of surrounding valve tissue in the order valve – style – stigma.In the absence of valve tissue, as in ett-1 gynoecia, a completebasalization occurs, and the majority of the gynoecium appearsto be composed of style tissue. This is suggested by the appear-ance of style-like epidermal cells along the stalked and lowerovary regions of ett-1 gynoecia, and of style-specific reportergene expression in the upper ovary part of ett-1 gynoecia. Thelatter represents both a basalized and an inverted developmentof tissues (see below).

Inversion of the transmitting tract in ett gynoeciaThe inversion of identity in the inside-outside dimension issuggested by the expansion of functional transmitting tract in theabaxial direction (indicated by blue in Fig. 13). The similaritiesof the abaxial outgrowths and the normal transmitting tractinclude the spongy arrangement and staining of subepidermalcells with alcian blue and their ability to serve as functional trans-mitting tract for wild-type pollen. The inversion is also suggestedby the appearance of ASA1:GUS expression in the external celllayers of ett surface outgrowths. ASA1:GUS expression in thewild-type style does not occur in the outer epidermis, and isrestricted to the cells of and surrounding the transmitting tract andthe postgenitally fused inner epidermis. Thus the ASA1:GUSexpression in ett gynoecia suggests that the inverted transmittingtract tissue is more style-like than ovary-like, as the transmittingtract in the wild-type ovary does not exhibit ASA1:GUS activity.GUS expression in the novel unidentifiable cells of the outerepidermis of ett-3 and ett-1 gynoecia suggests that these cellscould have an identity similar to the cells of the inner (normallyfused) epidermis of the style. These novel cells also induce pollengermination. That decapitated styles will induce pollen togerminate (Kandasamy et al., 1994) supports the hypothesis thatthe novel outer epidermis cells in ett gynoecia are similar to wild-type inner epidermal style cells.

Possible roles for ETT functionThese results suggest that ETT function is necessary for thecoordination of positional information (Wolpert, 1969) alongthe longitudinal and transverse axes of the developing Ara-bidopsis gynoecium. It is clear that in the absence of completeETT function, the correct development of cell types in theirnormal apical-basal and inside-outside order does not occur.The relationship between the resultant abnormalities of basal-ization of characters, abaxial expansion of transmitting tract,ovary decrease and internode elongation in ett gynoecia,however, is unclear. For example, it is unclear how one geneticdeficiency results in both valve decrease and abaxial expansionof transmitting tract, or if it affects one of these processesdirectly, and the other secondarily. Likewise, it is unknown ifthe alterations in vascular patterning are causal or resultant ofthe alterations in apical-basal patterning.

Nevertheless, some aspects of ett phenotypes suggestpossible patterning events that occur during wild-typegynoecium development, as well as roles for ETT functioningin these events. It is known that ett gynoecia primordia aremisshapen at the very earlieast stages of development (Alvarez,1994; A. S. unpublished data). This suggests that ETT functionsvery early in the formation or elaboration of a prepattern spec-ifying apical-basal and adaxial-abaxial patterning in thegynoecium primordium. The stochastic formation of valvetissue on the two lateral sides of an ett gynoecium suggests thatthe two sides develop independently, or that ETT function is

necessary for their coordination. Lastly, the composition of sto-chastically forming sectors of valve tissue abutting style cellsabutting stigmatic cells, suggests that a positional informationmechanism exists for juxtaposing these cell types, and that ETTis involved in polarizing this information in one direction.

Besides its role in global patterning, the data suggest that ETTfunction is necessary for the establishment of fields of valvetissue on the lateral sides of developing gynoecia. The mostseverely affected allele, ett-1, results in the complete absenceof valve tissue. Double mutant analyses of ett-1 with homeoticmutations (ap2-2 and pi-1) that position carpel (valve) tissue inthe first and third whorls of the flower indicate that valve tissueformation is only affected in the fourth whorl by ett-1(A.S.unpublished data). Thus, ETT appears to be involved in valvetissue formation only in the fourth whorl, and this function isnot required for the differentiation of valve tissue per se. Thissuggests that ETT function is involved in the positioning ofvalve cell types rather than in their molecular differentiation.

ETT participation in valve formation may be causative orrestrictive. For example, ETT may act in the recruitment ofcells to form a field of valve tissue. Alternatively, ETT mayparticipate in positioning a style-valve boundary in the devel-oping gynoecium, since this boundary is retained in ett-2 andett-3 gynoecia, but shifted relative to the normal position. Themispositioning of this boundary could affect the potential sizeand position of a valve sector. In addition, ETT may beinvolved in establishing the polarity, or direction in whichpositional information is interpreted relative to boundarypositions. This could explain the circular style-valve boundaryin ett-3 gynoecia, as well as the reversal of polarity of theoutside surface of ett gynoecia. The cloning of the ETT locusand the localization of its gene product during developmentmight distinguish between these possibilities.

Finally, some features of the ett phenotypes are not novel tobotanists. The basal solid region of the internode-like structurefound inbetween the ovary and the medial stamens of ettflowers is found in other members of the Brassicaceae whereit is referred to as a stipe or gynophore. Likewise, the growthof stigmatic tissue at carpel margins (if the two carpel modelis to be believed for Arabidopsis and the Brassicaceae) is notunheard of and may represent an ancestral condition of theangiosperms (Baily and Swamy, 1951).

We are indebted to Ken Feldman for generation of the ett-1 and ett-2 alleles, to John Alvarez and David Smyth for the isolation and giftof the ett-3 to ett-8 alleles, and to Kris Niyogi and G. Fink for the giftof the ASA1:GUS lines. We are also indebted to Judy Roe and DonKaplan for help with all aspects of this work. We thank Tim Durfee,Sarah Hake, Fred Hempel, and Chad Nusbaum for critical commentson versions of this manuscript, members of the Zambryski lab forgeneral assistance, Wilfred Bentham, Don Pardoe, and the Berkeleyelectron microscope facility for help with SEM, and Steve Ruzin andthe NSF Center for Plant Developmental Biology for assistance withmicroscopy. A. S. thanks Miko Maruoka and Toshi Foster for encour-agement and advice. This work was supported by Department ofEnergy grant 88ER13882 to P. C. Z.

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(Accepted 13 January 1995)

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Arabidopsisgynoecium structure in the wild type and in … · compare the wild-type pattern of gynoecium cell types and regions, with patterns formed in gynoecia of individuals homozygous