Arabidopsis gynoecium structure in the wild type and in ettin mutants

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1 Development 121, (1995) Printed in Great Britain The Company of Biologists Limited 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 ( SUMMARY The gynoecium is the female reproductive structure of flowering plants. Here we present a description of the Arabidopsis thaliana gynoecium at anthesis. The cylindrical organ can be broken down into three longitudinal regions arranged in an apical-basal order: stigma, style, and ovary. Each region can be distinguished histologically and morphologically. The transmitting (pollination) tract is axially positioned along the center of the gynoecium and spans stigma, style and ovary. Histochemistry, scanning electron microscopy and a style-specific reporter gene are used to compare the wild-type pattern of gynoecium cell types and regions, with patterns formed in gynoecia of individuals homozygous for a series of allelic mutations at the ETTIN locus. ettin gynoecia show morphological and histological alterations that appear to result from the merging of apical and basal regions and the development of abaxial into adaxial tissues. These developmental abnormalities result in a reduction of the ovary region, an expansion of the stylar and stigmatic regions, and the abaxial (outward) proliferation of transmitting tract tissue. The alterations in the mutants can be interpreted as resulting from misspecifications of position along the longitudinal and transverse axes during gynoecium development. The results suggest that early patterning events underlie wild-type gynoecium development, and that ETT functions during this early programming. Key words: Arabidopsis, gynoecium, ETTIN, positional information, flower development INTRODUCTION While our understanding of the molecular genetics governing Arabidopsis floral meristem and organ identity has increased (recently reviewed by Okamuro et al., 1993; Weigel and Meyerowitz, 1994), regional specification within individual floral organ types has remained largely unexplored. Many flowers 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 the gynoecium. The reproductive organs exhibit distinct regional differences in shape and composition along their length, making them useful in genetic analyses of development at the suborgan level. The gynoecium is the female reproductive organ system and is defined as the collective whorl(s) of carpels, or megaspore bearing leaves, that terminate all fruit bearing flowers (reviewed by Gasser and Robinson-Beers, 1993). Along their longitudinal axis, most gynoecia are composed of three distinct regions: a basal ovary, a style, and an apical stigma. The transmitting (pollination) tract and the ovule producing placenta are two additional gynoecium-specific regions. The transmitting tract is a contiguous tissue spanning the entire structure, and is composed of specialized cells that guide the growth of pollen tubes. Within the ovary, the pollen tubes deliver sperm to the egg cell-containing ovules, which facilitate fertilization and enclose the developing embryos. Most of the cells that make up these five regions are functionally and/or structurally distinguishable. Several mutant loci have been reported that alter gynoecium development 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) and spatula (Alvarez and Smyth, 1994a). The interpretation of these mutant phenotypes and of their genetic relationships however, is currently limited by the lack of a cell and tissue level description of the wild-type gynoecium. Here we describe the overall structure of the sexually mature Arabidopsis gynoecium excluding the ovules, which have already been described (Mansfield et al., 1991; Robinson- Beers and Gasser, 1992). We also describe the phenotypes produced in homozygotes of members of an allelic series of mutations at the ETTIN (ETT) locus. These results suggest that a spatial prepattern is programmed in the gynoecium primordium and that ETT functions in its establishment and/or elaboration. MATERIAL AND METHODS Genetic materials ett-1 and ett-2 were isolated from T-DNA mutagenized seeds of the Wassilewskija (WsO) ecotype (Feldmann, 1991). ett-3 was isolated by John Alvarez and David Smyth from a population of ethylmethane

2 1520 R. A. Sessions and P. C. Zambryski sulfonate (EMS)-treated Landsberg (LaO) seeds. These three mutations were shown to not complement in the three pairwise F 1 (Fig. 5E) and F 2 combinations, and to be linked to the erecta (er) locus on chromosome 2 (not shown). ett-1 has been mapped very near the asymmetric leaves (as) locus on chromosome 2 based on the following results: plants homozygous for er, as and ett-1 were crossed to plants heterozygous for the same mutant loci, in cis. Of the 90 F 1 progeny, 48 were wild-type, 35 were er, as, ett-1, 4 were er, and 3 were as, ett-1. No recombinants were found between ett-1 and as. ett-1 is not allelic with pinoid mutations, which reside at a locus near as (not shown). Wild-type plants described here are of the WsO ecotype. All plants were grown in Sunshine Mix soil, in 22 C growth chambers, under 18-hour-light days, and watered with Miracle Grow plant food. Thin section histology Tissue was fixed in FAA (50% ethanol, 5% glacial acetic acid, 10% formalin, 1% Triton X-100). Tissue was then dehydrated in ethanol and embedded in JB-4 or JB-4 Plus (Polysciences Inc) plastic resin. Sections were cut to 3 µm and adhered to glass slides using water and a slide warmer. Slides were stained in one of three ways. (i) Periodic-acid-Schiff s (PAS) reaction followed by toluidine blue (TBO) (Feder and O Brien, 1968). Slides were air dried and mounted with Permount and a cover slip. Carbohydrates stain a deep pink to red, and other components are stained blue with TBO. (ii) Alcian blue (Pearse, 1980) followed by neutral red (NR). NR staining was performed in a 1% aqueous solution for 15 seconds. NR was destained with distilled water. Slides were air dried and mounted with Permount and a cover slip. Acidic polysaccharides stain a deep blue. (iii) 0.1% aniline blue in 0.1 M potassium phosphate buffer (ph 7.5 or 9), with 40% glycerol and 0.1% n-propyl gallate (fluoresence extender) for >30 minutes and mounted in the same solution with a cover slip. Viewed with fluorescence optics (Zeiss axiophot compound light microscope) callose in pollen tubes and phloem elements stains at ph and appears white and the cuticle of the style epidermis stains at ph 9 and appears orange. Whole-mount staining Gynoecia were cleared and stained using the same method to reveal both the vasculature (xylem) and pollen tubes. Gynoecia viewed to assess pollen tube growth were emasculated before anthesis, manually pollinated with WsO pollen, and allowed to grow for 48 hours. Gynoecia were dissected out of flowers, fixed in FAA for 24 hours, washed in 0.1 M potassium phosphate buffer (ph 7.5) for 2 hours, soaked in 8 M NaOH for 12 hours, rewashed in buffer for 1 hour, and stained in 0.1% aniline blue in the same buffer for 30 minutes to 2 hours. Viewed with visible light, 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 anthesis stage 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 with PAS and TBO. Elongated stigmatic cells are indicated by arrowheads. (E) Cross section through an anthesis stage style stained with PAS and TBO. Transmitting tract (t), fans of xylem elements 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 the epidermis), pollen tubes (pt). (F) Cross section through an anthesis stage ovary stained with PAS 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 is indicated by an asterisk. The regions of valve senescence are indicated by arrowheads (only for the left side of this ovary) and define the limits of a valve. (G) The two carpel model. Scale bar in lower left corner: (B) 1 mm, (C) 150 µm, (D) 22 µm, (E) 35 µm, (F) 52 µm.

3 Arabidopsis gynoecium structure 1521 the xylem in these gynoecia stains a dark blue. Viewed with fluorescence optics, the pollen tubes in these gynoecia fluoresce bright white. Scanning electron microscopy (SEM) SEM was performed according to Roe et al., GUS assays The ASA1:GUS containing line was provided Kris Niyogi and G. Fink. Assays for GUS activity in ASA1:GUS containing plants were performed in 50 mm sodium phosphate (ph 7), 0.05% Triton X-100, 0.1 mm K 3Fe(CN) 6, 0.1 mm K 4Fe(CN) 6, and 1 mm X-glucuronic acid, at 37 C for 12 hours, after an initial 10 minutes vacuum infiltration of the assay solution. ASA1:GUS containing plants express GUS at high levels in the style in unemasculated, post anthesis flowers, and at lower levels on the floral peduncle (Niyogi and Fink, in preparation). In our hands style-specific ASA1:GUS reporter gene expression is only activated in the presence of pollen (i.e. pollen inducible). Emasculated flowers from ASA1:GUS plants never stain for GUS activity in the style (not shown). Pollen from ASA1:GUS plants does not express GUS activity when grown on wild-type plants (i.e., the GUS expression in the style of ASA1:GUS self pollinated plants is not from the pollen tubes themselves). Of our GUS assays on ett, ASA1:GUS plants, we performed approximately half on emasculated flowers that had been pollinated with wild-type pollen, and half on self pollinated flowers. Since this reporter is pollen inducible we tried to cover each gynoecium that we assayed with as much pollen as possible. We also viewed >50 gynoecia of each genotype assayed (>200 total) to see all the possible GUS expressing tissues. Representative staining patterns are presented in Fig. 11. ASA1:GUS is also wound 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 significant relative to the style tissue expression. RESULTS General structure of the wild-type gynoecium The Arabidopsis flower is terminated by the gynoecium (Fig. 1A). The early development of the Arabidopsis gynoecium involves the formation of a cylinder from the region of the floral meristem above the medial stamen (Smyth et al., 1990). Later development involves the postgenital fusion of the inner surfaces and tip of this cylinder, and the development of ovules along the margins of the fused walls, to form a closed bilocular chamber (Hill and Lord 1989; not shown; for an example of a similar postgenital fusion in the Brassicaceae see Boeke, 1971). After anthesis and fertilization of the egg cells, the gynoecium elongates approximately sevenfold into the mature seed containing silique, or fruit. The silique then dehisces liberating the seeds and the valves (Fig. 1B). The septum is the postgenitally fused partition and remains on the plant after dehiscence. Fig. 2. Idioblast position and valve organization. (A) Cleared valve from 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 in the lateral plane stained with PAS and TBO, showing a lateral vascular 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 through an anthesis stage valve in the lateral plane stained with PAS and TBO. Intermediate bundle (i) in cross section, chlorenchyma (cl). (D) Cross section through an anthesis stage valve stained with PAS and TBO. Lateral vascular bundle (l) and the associated idioblasts (id). (E) Paradermal longitudinal section through the top of an anthesis stage gynoecium in the lateral plane stained with PAS and TBO. Idioblasts (id) develop along the medial vascular bundle (m) at the junction of the ovary (o) and the style (st). Scale bar in lower left corner: (A) 166 µm, (B) 22 µm, (C) 19 µm, (D) 19 µm, (E) 38 µm. Stigma The stigma is the epidermis of the distal gynoecium and is composed of approximately 150, bulbous, elongated epidermal cells that are specialized for pollen attraction and recognition (Fig. 1C, D). The stigma is the only epidermis that will induce pollen germination on an anthesis stage gynoecium (Kandasamy et al., 1994). Following germination, pollen tubes grow intrusively between the papillar cells into the center of the style (Kandasamy et al., 1994). The stigma is the beginning of the transmitting tract. Style The solid style is a postgenitally fused cylinder composed of cells specialized for secretion. Polysaccharides are abundant at its distal epidermis (the stigma) and within its axial core as revealed by staining with PAS (Fig. 1C) and alcian blue (see below). PAS and alcian blue stain polysaccharides which are localized in the transmitting tract of another member of the Brassicaceae (Hill and Lord, 1987). In Arabidopsis the routes of pollen tubes are tightly correlated with the presence of the PAS and alcian blue staining regions (see below). The stylar core is composed of axially elongated cells between which the pollen tubes grow (Figs 1E, 3A; Kandasamy et al, 1994), and is 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; and see below).

4 1522 R. A. Sessions and P. C. Zambryski Ovary The ovary is a longitudinally bissected cylinder as seen in cross section in Fig. 1F and is partitioned by the septum. The axial center of the septum is the continuation of the transmitting tract. The apoplasm of these inner septal cells reacts with the PAS (Fig. 1C,F) and alcian blue polysaccharide stains, and the pollen tube detecting callose stain aniline blue (see below). The postgenital fusion plane in the septum is denoted by an asterisk and an arrow in Fig. 1F. Alongside the septum are positioned approximately 45 ovules in four rows. The position of the senescent zones that will participate in fruit dehiscence (and define 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 Arabidopsis gynoecium is common for many members of the Brassicaceae, whose gynoecia are thought to be composed of two congenitally fused carpels that develop in the lateral plane of the flower (reviewed by Okada et al., 1989). According to the two carpel model (Fig. 1G), the valves represent the carpels, and the septum, style and stigma represent their postgenitally fused margins and remaining submarginal placentae. With regard to floral symmetry the gynoecium is oriented in the flower with the septum in the medial plane (Fig. 1F,G). All cross sections in this paper, unless noted, are oriented with the medial plane positioned vertically. Gynoecium cell and tissue types Certain cells and tissues have properties that distinguish them from other cells and tissues. These distinguishing features are useful in comparative analyses of positional differences. The cell types described here pertain to their appearance and position at anthesis (stage 13; Smyth et al., 1990). (a) Idioblasts One distinguishable cell type is a large and elongate, phloem associated idioblast found in relatively invariant positions in the flower. There are four main vascular bundles that supply the gynoecium (Fig. 1F). In the ovary, the idioblasts are only associated with bundles that supply the valves. A cleared valve from a dehiscent silique is shown in Fig. 2A and stained to reveal the patterning of the lateral and intermediate bundles. An anthesis stage gynoecium is shown in longitudinal (Fig. 2B,C) and cross (Fig. 2D) section to reveal the presence of the idioblasts in relation to the lateral and intermediate bundles. The idioblasts are only associated with the medial bundles above the ovary at the junction of the style. Fig. 2E shows a paradermal section spanning the junction of the style and ovary demonstrating the distinctive appearance of these idioblasts in PAS stained sections. The idioblasts in the ovary generally stain with a more granular appearance (Fig. 2B) than those in the style (Fig. 2E). The idioblasts described here may be 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 vascular bundle. In areas lacking a bundle (Fig. 2C,D) there are 4 morphologically distinct cell types proceeding from outside in: an outer epidermis, 3 layers of chlorenchyma, a longitudinally elongated layer subadjacent to the inner epidermis (asterisk), and the radially elongated inner epidermis. The vascular Fig. 3. Transmitting tract tissue. (A) Medial longitudinal section through pollinated gynoecium stained with alcian blue and neutral red (NR). The transmitting tract stains blue, pollen tubes (pt) stain a deep red. Pollen grain (pg), stylar xylem (sx). (B) Serial section of A stained with aniline blue, at ph 7.5, and viewed with fluorescent light. Pollen tube (pt) walls and callose plugs (cp) fluoresce white. Stylar xylem (sx) is autofluorescent. (C) Alcian blue and NR stained medial longitudinal section through the septum (sp) in the ovary showing the transmitting tract (t). The postgenital fusion plane in the septum is indicated with an asterisk. (D) Cross section through the ovary stained with alcian blue and NR. Transmitting tract (t), postgenital fusion plane (*), ovules (ov). Scale bar in lower left corner: (A and B) 38 µm, (C) and (D) 19 µm.

5 Arabidopsis gynoecium structure 1523 bundles take the place of the innermost chlorenchyma layer in the areas in which they develop (Fig. 2B-D). Fig. 4. Scanning electron micrographs and thin section histology of epidermal tissue types. (A) Top of an anthesis stage gynoecium showing the stigma (sg), style(st) and valve (v) surfaces. Pollen grains (pg). (B) The style (st) epidermis is composed of interlocking cells with crenelated wax deposits, and stomata (s). (C) Cross section through the style stained with aniline blue ph 9. The crenelated wax cuticle exhibits a distinct fluoresence when stained by basic solutions of aniline blue. The stylar xylem (sx) autofluoresces. (D) Surface of the medial side of the ovary. A furrow (large arrow) runs along the medial surface and separates the valves (v), only one of which is shown here. The valves are covered in a mosaic of 4-5 cell clusters that contain immature unopened stomata (small arrows) at their centers. (E) Gynoecium base showing the medial surface. The surface between the valves (v) is composed of cuboidial cells and stomata. 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) 22 µm, (C) 19 µm, (D) 27 µm, (E) 28 µm. (c) Transmitting tract The transmitting tract is distinguishable on the basis of its alcian blue staining (Fig. 3A,C,D) and its capacity for pollen tube growth (as revealed by aniline blue in Fig. 3B). Aniline blue staining reveals the callose in the pollen tube walls, and in the plugs that are laid down proximally as the tip grows (Fig. 3B). The transmitting tract cells from style and septum can be distinguished on the basis of their arrangement. In the septum they are positioned adjacent to cells arranged in a spongy architecture separated by air spaces (Fig. 3C), whereas in the style they are tightly packed and more elongate (Fig. 3A). The transmitting tract is 4-5 cells thick under the inner epidermis (Fig. 3C,D), as seen in the septum where the epidermis does not bind alcian blue. The postgenitally fused inner epidermis in the style however, is not obvious, and binds alcian blue (Fig. 3A, see also 8E). (d) Epidermal cells The stigma, style and ovary each have a distinct epidermis. Stigmatic cells are characteristically elongate, and bulbous towards the base (Fig. 4A). The epidermis of the style is distinguishable from that of the ovary and stigma by the presence of open stomates and crenelated wax deposits (Fig. 4B) that fluoresce when stained by aniline blue in basic ph (Fig. 4C). In the ovary, the valve epidermis is distinguishable from that of the intervalve region (Fig. 4D). The valve epidermis is a patchwork of 4-5 cell clusters that surround an immature, unopened stomata (Fig. 4D). These stomata open during fruit development (not shown). The intervalve region is furrowed and covered by small cuboidal cells (Fig. 4D) except at the very base of the gynoecium, where this surface protrudes out and contains stomata in its epidermis (Fig. 4E). ettin mutations The ETTIN locus is currently defined by ten recessive noncomplementing mutant alleles (see Materials and Methods). Structural alterations in the gynoecium are the most apparent phenotypic consequences of the strong ett-1 mutation on floral form (compare Fig. 5A and D) and include: split styles and stigmas, a decrease in ovary size, a reduction in septum formation, tapering of the gynoecium into a stalked like structure, and outgrowths of tissue from the ovary in the medial plane. Defects in the first three organ whorls also occur in approximately 50% of ett-1 flowers and include the production of additional sepals and petals, and some anther loss (not shown). In this paper we focus on the gynoecial abnormalities produced by three representative alleles: ett-1, ett-2, and ett-3 (Fig. 5B-D). Individuals heteroallelic for the three pairwise combinations of ett-1, ett-2 and ett-3 were constructed to determine relative allele strength. Using silique length as a measure of ovary size, and hence an estimate of gene function (see below), Fig. 5E demonstrates that certain alleles will provide some phenotypic rescue in trans to other alleles. The ett-2 allele provides some phenotypic rescue to the ett-1 and ett-3 alleles (compare lengths 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 rescue to ett-1 (Fig. 5E). The ett-1 allele has yet to provide in trans phenotypic rescue to any other allele (not shown), and may represent a null mutation. Thus, the order of increasing allele strength is ett-2<ett-3<ett-1. Fertility differences follow the allelic series: ett-2 homozygotes are fertile, ett-3 homozygotes are slightly fertile, and ett-1 homozygotes are usually sterile. The differences between wild-type and ett gynoecia can be broken down into: (i) alterations in vascular patterning, (ii) a reduction in ovary size and an outgrowth of transmitting tract in the medial plane, and (iii) the appearance of stylar and stigmatic chacteristics in the ovary region. These alterations will be considered individually. Additional differences between wild-type and ett gynoecia that will not be considered in detail here include the elongated internode, or stalk, that develops beneath ett ovaries, and female sterility.

6 1524 R. A. Sessions and P. C. Zambryski Fig. 5. Scanning electron micrographs of ettin alleles and the relationship between allele strength and valve size. Valve extent is indicated for the right side of each gynoecium as the space between the two arrows. (A)-(D) Post pollination gynoecia homozygous for ett alleles. (E) Stage 18 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 gynoecia Whole-mount staining reveals the four main bundles of the wild-type vascular system (Fig. 6A). The bundles in the lateral plane, which branch to supply the valves (Fig. 2A), terminate just below the style (Fig. 6A). The medial bundles, which supply the placentae, enter the style, and bifurcate into fans of xylem 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 between medial and lateral bundles (see Fig. 2). Fig. 6. Vascular patterning in cleared, whole-mount anthesis stage wild-type and ett gynoecia. For each gynoecium the medial (m) and lateral (l) vascular bundles are labeled, as is the stylar xylem (sx) that terminates the medial bundles. The point along the medial bundle at which the bifurcation occurs is indicated by an asterisk. The limits of the ovule containing ovary are also indicated in each gynoecium by two large arrows. (A) Wild-type gynoecium viewed medially. Lateral bundle termination and medial bundle bifurcation (*) occur at the distal limit of the ovary. Only one fan of stylar xylem (sx) is seen in this view. (B) ett-2 gynoecium viewed laterally. Lateral bundle termination and medial bundle bifurcation (*), occur below the distal limit of the ovary (the two branches of a single medial bundle are not seen in this view). Medial bundle bifurcations anastamose with bifurcations of the other medial bundle in the stylar xylem (sx). (C) ett-3 gynoecium viewed medially. Medial bundle bifurcation is basalized. Lateral bundles have bifurcated, and terminate in stylar xylem-like clusters (sx ). Valve (v). (D) ett-1 gynoecium viewed medially. Notice anastomosed medial bundle bifurcation branches. Scale bar in lower left corner: (A) 200 µm, (B-D) 166 µm.

7 Arabidopsis gynoecium structure 1525 The vasculature of ett gynoecia shows alterations in apicalbasal patterning, and a mixing of medial and lateral characters in the individual bundles. Apical-basal patterning defects include the mispositioning (relative to the distal limit of the ovary) of lateral bundle termination and of medial bundle bifurcation (Fig. 6B-D). The mixing of lateral and medial bundle characters occurs in lateral bundles that bifurcate and terminate in medial bundle-like fans of xylem (Fig. 6C). The severity of the alterations does not parallel the allelic series. Instead, the vascular patterns produced by the weak and strong alleles are more similar to each other than to the intermediate allele. ett-2 (Fig. 6B) and ett-1 (Fig. 6D) gynoecia show a similar basalizing of the vasculature with medial bundles splitting and lateral bundles terminating below the distal limits of the ovary. Unlike the wild type, the bifurcation branches of each medial bundle in ett gynoecia enter the lateral sides of the ovary and anastomose with the branches of the other medial bundle in the style (Fig. 6B-D). Consistent with allele strength designations, these alterations are more severe in ett-1 gynoecia (compare Fig. 6B and D). The medial and lateral bundles in intermediate strength ett- 3 gynoecia are phenotypically variable, and exhibit increased branching compared to the wild type. Regarding the basalized medial bundle bifurcation, and medial bundle anastamoses, ett- 3 gynoecium vasculature patterning is similar to that produced by the weak and strong ett alleles (Fig. 6C). ett-3 medial bundles differ in the variable occurance of branches in the ovary region. These branches terminate in style-like arrays that are found in the distal limits of the ovary (Fig. 6C). The ett-3 lateral bundles are phenotypically variable and exhibit bifurcations, basalized termination and/or termination in a styletype fanned array of xylem. For example, Fig. 6C shows a gynoecium in which both lateral bundles have bifurcated and terminated in stylar type arrays that are positioned in the medial outgrowths. The termination of a lateral bundle in a stylar-like array is a character normally associated only with medial bundles. One consistent alteration in vascular patterning observed in the three alleles is the association of densely staining idioblasts along the entire length of the medial bundles (Fig. 7B-D). Idioblasts with this appearance are normally found at the styleovary junction along the medial bundles (Fig. 2E), and are absent from the medial bundles in the ovary region (Fig. 7A). As densely staining idioblasts are only associated with the medial bundles at the style-ovary junction in the wild-type gynoecium, the altered association in ett mutants suggests that ett medial tissues in the ovary are more stylar in composition. In summary, the vasculature of ett gynoecia exhibits a general trend towards basalization of pattern. This is suggested by the basalized (relative to the distal limits of the ovary) expression of lateral bundle termination, medial bundle bifurcation, and idioblast association with the medial bundles. (ii) Reduction in ovary size and abaxial development of transmitting tract in ett gynoecia As was seen in Fig. 5, as ETT function decreases, the size of the ovary decreases. Although the ovary decrease is most apparent as a reduction of valve tissue (Figs 5, 8A-D), the septum (Fig. 8C,D,L) and placentae (see below) are also reduced. In the apparent place of the missing ovary is seen the development of everted transmitting tract tissue (Fig. 8C,D,J- L), an internode-like (stalked) structure (Fig. 8B-D) and style tissue (see below). The everted transmitting tract tissue in ett gynoecia forms in and on outgrowths above (Fig. 8F-H), between (Fig. 8J-K) and below (Fig. 8C) sectors of valve tissue, as well as in the apparent place of missing valve tissue (Fig. 8D,H and L), in an allele strength-dependent manner. This outwardly facing transmitting tract is similar to that of the wild type (Fig. 3C) in being 4-5 cells thick under an epidermis (Fig. 8E-L). That these outwardly facing alcian blue-positive tissues can serve as functional transmitting tract is demonstrated by pollinating ett gynoecia with wild-type pollen (Fig. 9B-D). Wild-type pollen germinates 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. The medial outgrowths (mo) contain idioblasts (id), and transmitting tract (t ) at their periphery (see Fig. 8). (A) Wild-type, showing the absence of idioblasts along the medial bundle in the ovary. Medial bundle (m), septum (sp), transmitting tract (t), valve (v), xylem (x). (B) ett-2, showing numerous 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 the medial bundle (m). A pollen grain (p) has landed on the medial outgrowth and germinated. (D) ett-1 showing the absence of valves and the presence 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.

8 1526 R. A. Sessions and P. C. Zambryski

9 Arabidopsis gynoecium structure 1527 Fig. 9. Pollen tube routes in wild type and ett mutants. Whole-mount gynoecia pollinated with wild-type pollen (p), stained with aniline blue and 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), which is 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 upper ovary of ett-1 gynoecia (Fig. 9D). The epidermis of these tissues cover alcian blue staining subepidermal tissues through which the pollen tubes grow (compare Figs 8 and 9). The stalked regions of ett gynoecia, although uniform in surface view (Fig. 5B-D), are not uniform internally (Fig. 8B- D). For example, the stalks in ett-1 gynoecia are composed of a basal region of solid stem (in), a middle region of solid stem formed by a post genital fusion event (in ), and an apical region 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 and NR to reveal the transmitting tract (t). Transmitting tract tissues stain blue. With increasing allele strength ett gynoecia undergo the development of transmitting tract (t ) and a stalk (in, in, in in place of valve tissue (delimited by arrowheads to the right of the gynoecia in A-C). Weak alleles result in the outgrowth of this tissue in the medial plane of the gynoecium (mo), whereas intermediate and strong alleles result in its development in the lateral plane (lo) as well. (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-type gynoecium. Transmitting tract (t), internode (in). (B) ett-2 gynoecium with expanded internode, and shortened valve. (C) ett-3 gynoecium showing reduced valves, lateral outgrowths (lo) containing transmitting tract (t ), and a basal stalk. Although uniform in surface view (see Fig. 5B), this stalk is composed of a lower solid stem (in) and an upper region containing ovules (in ). (D) ett-1 gynoecium lacking valves. Transmitting tract tissue (t ) develops in their place. The lower stalk, although uniform in surface view (see Fig. 5D), is tripartite: a solid basal region (in), a middle solid region that has resulted from a postgenital fusion event (in ), and an upper region containing ovules (in ). (E-L) Cross sections at levels indicated in (A-D). With increasing allele strength is seen the development of transmitting tract tissue (t ) in the medial plane, and its expansion to the lateral sides of the gynoecium. (E-H) Transmitting tract proliferation (t ) in the style is accompanied by a basalized expression of stigmatic tissue (sg ). (E) Wild-type, (F) ett- 2, (G) ett-3, (H) ett-1. (I-J) Transmitting tract proliferation in the ovary. 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 other ett gynoecia is allele strength dependent (Fig. 8B,C). A decrease in ovule production also parallels increases in allele strength. On average (n=10), wild-type WsO and LaO gynoecia have 47 (±4.4) ovules, ett-2 gynoecia have 32 (±3.2) ovules, ett-3 gynoecia have 15 (±3.4) ovules, and ett-1 gynoecia have 14 (± 3.9) ovules. The ovules in ett-2 gynoecia appear morphologically normal and are fully fertile (not shown). Ovules in ett-3 and ett-1 gynoecia are almost completely sterile and are abnormal (not shown). In summary, all aspects of the ovary of ett gynoecia show decreases in size. Unlike the septum and the placentae, the decrease in valve formation can be complete. The appearance of transmitting tract in the outer layers of ett gynoecia suggests that normal abaxial-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 ett gynoecia (a) Epidermal characters The reduction in the ovary regions of ett gynoecia is coupled with a basalization of stigmatic and style-like characters. Style and stigma epidermal cell types are found on the surface outgrowths of ett gynoecia. These include style-like cells along the flanks of the medial outgrowths of ett-2 (Fig. 10A,B), and ett-3 (Fig. 10C, surrounding the valves) gynoecia, and along the stalked surfaces of ett-3 and ett-1 gynoecia (Fig. 10D,F,G). Stigmatic cells are found along the medial outgrowths of ett-1 and ett-3 gynoecia (Fig. 10C,E). Additionally, an unidentifiable novel, cell type (asterisk) develops between and beneath (Fig. 10C) the valves in ett-3 gynoecia, and between the apical stigma and basal stalk in ett-1 gynoecia (Fig. 10E). These are the cells that cover the alcian blue staining tissues in ett gynoecia and that facilitate wild-type pollen germination. In ett-3 and ett-1 gynoecia they form sharp boundaries with the style-like cells on the stalks beneath them (Fig. 10C,F,G). (b) Style-specific reporter gene expression Another character found to exhibit basalized expression in ett gynoecia is the β-glucuronidase (GUS) reporter gene driven by the Anthranilate Synthetase 1 (ASA1) promoter. In wild-type

10 1528 R. A. Sessions and P. C. Zambryski 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-2 gynoecium 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. A small 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 medial outgrowth (mo). Below the valve sector are cells of unknown type (asterisk). The epidermis of the stalked region is composed of cells with crenelated 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-3 gynoecium 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 of the 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 medial outgrowth cells are smooth and bulbous, whereas those of the stalk have crenelated wax deposits and interlocking shapes like the style epidermis. There is a noticable boundary around the gynoecium at this level (arrow) between the two cell types. (G) Longitudinal section in the medial plane of an ett-1 gynoecium at the level of the boundary (arrow) indicated in Fig. F, stained with aniline blue and viewed with fluorescent light. Cells below the boundary (arrow) have a cuticle that fluoresces like that of the style epidermis (arrowhead). Scale bar in lower left 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 the inner layers of the post anthesis stage style (Fig. 11A,B; K. Niyogi and G. Fink, in preparation; see Materials and methods). ett plants containing the ASA1:GUS sequences show strong GUS activity in more basal regions of their gynoecia. The tissues showing basalized GUS expression include those of 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 adaxial transmitting tract, ASA1:GUS expression is found in the outer layers of the surface outgrowths of ett gynoecia (Fig. 11D,F,H). In contrast to the basalized style epidermis in ett gynoecia, ASA1:GUS expression does not extend along the entire length of either ett-2 medial outgrowths (Fig. 11C) or the ett-3 (Fig. 11E) and ett-1 (Fig. 11G) stalked regions. It is possible that only the epidermal surfaces, and not the internal layers of the lower medial outgrowths of ett-2 gynoecia and the stalks of ett-3 and ett-1 gynoecia, exhibit the basalized expression of style characters. ettin phenotypic variability The ett phenotypes presented are those found in the highest proportions. There is however some phenotypic variability. This variability is most obvious in the differential expression of valve tissue along the two lateral sides of the gynoecium. In all three alleles, each lateral side of the gynoecium has the potential to develop into a tissue that is different from that of

11 Arabidopsis gynoecium structure 1529 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-2 gynoecium 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 plane runs horizontally across the figure. The cuticle of the style-like (st ) side of the gynoecium fluoresces (arrowhead). Pollen tubes (arrows) grow in the medial outgrowths (mo) and within the septum. (D) SEM of an ett-3 gynoecium in which no valve tissue has developed on the lateral side. 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 of valve (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.

12 1530 R. A. Sessions and P. C. Zambryski 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) ett-3 (n=150) ett-1 (n=40) *The amount of valve tissue on the two sides of a gynoecium is rarely the same. **n= total number of flowers observed from three different plants. the other side. Thus, the two lateral sides of the gynoecium develop independently. This is seen subtly in Fig. 5B and C as a difference in the length of valve tissue that has formed on the two 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 that does not express the style-specific GUS reporter gene. This variability is summarized in Table 1 and in Fig. 12 and is highest for the intermediate strength allele. Whereas ett-2 and ett-3 gynoecia usually have valve tissue, ett-1 gynoecia rarely do. In ett-2 and ett-3 gynoecia that have valve tissue on only one lateral side, the other side develops into tissue that is style-like (Fig. 12A-D). The nonvalve side has style epidermal (Fig. 12B) and cuticular (Fig. 12C) features. When valve tissue does form in ett-1 gynoecia it is always surrounded by a ring of style and stigma epidermis (Fig. 12E) similar to the valve tissue in ett-3 gynoecia (Fig. 10C). The valve-style-stigma juxtaposition of cell types is similar to the wild-type condition, however the arrangement is radial in ett gynoecia whereas it is unidirectional in the wild type. basalization of those characters represented by yellow and blue in Fig. 13 in the apical-basal dimension, and an increased inversion of those internal characters represented by blue in the adaxial (inside)-abaxial (outside) dimension. The ovary reduction is easiest seen in Fig.13 as a decrease in the development 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 ett gynoecia Basalization is suggested by the appearance of stigmatic and stylar characteristics in the ovary regions of ettin gynoecia. These characteristics (indicated as blue papillae and yellow color in Fig. 13) include those of the epidermis, the vasculature, and style-specific reporter gene expression. Although each ett allele produces a different pattern of basalized features, the mispatterning of more apical features follows a general rule DISCUSSION We have described the anthesis stage Arabidopsis gynoecium. This structure is composed of at least 5 regions of distinguishable tissues: the stigma, style, valve, placentae and transmitting tract. We have used this description to analyze the phenotypes produced by a series of mutations at the ETTIN locus which result in alterations in the normal patterning of tissues in the developing gynoecium (Alvarez, 1994). The comparison of ett and wild-type gynoecia shows that a reduction in the ovary and a basalization of stigma- and stylelike tissues occurs as ETT function decreases. This suggests that ETT is involved in apical-basal gynoecium patterning that ensures the proper formation of the ovary and the restriction of stigma and style cell differentiation to the apical end of the developing gynoecium. Further, the abaxial development of the normally adaxially positioned transmitting tract in ett gynoecia suggests that ETT is also involved in the establishment of normal adaxial-abaxial polarity. The range of allele strengths and their resultant phenotypes suggest that ETT performs these functions in a dose dependent manner, rather than by a simple threshold effect mechanism. A color coded summary presented in Fig. 13 compares gynoecial organization between the wild type and ettin mutants and attempts to account for surface (top row) and internal (bottom rows) morphological and anatomical alterations that occur in gynoecia with decreasing amounts of ETT function (from left to right). In general, the ovary loss accompanying the decrease of ETT function is associated with progressive ETT = trans. tract = style = valve ett-2 =medial ovary = internode = idioblast = medial bundle Fig. 13. Summary of comparative analyses between wild-type and ett gynoecia. Major regions in the wild-type gynoecium and their rearrangement in gynoecia from plants homozygous for weak (ett-2), intermediate (ett-3), and strong (ett-1) ett alleles. This summary integrates information obtained using multiple markers and tissue characteristics, and attempts to simplify the data at hand. Each genotype is represented by a vertical column of three diagrams, except the wild-type, which has a fourth diagram representing the arrangement of tissues at the style-ovary boundary. Upper row represents surface views and lower rows represent sectional views at levels indicated by arrows in the upper row. The characteristics of cell types from five distinct regions of the wild-type gynoecium (leftmost column) are represented by five colors. Color usage as indicated. The transition states between more and less ETT function can be seen by proceding from left to right along any row. See text. ett-3 ett-1

13 Arabidopsis gynoecium structure 1531 of surrounding valve tissue in the order valve style stigma. In the absence of valve tissue, as in ett-1 gynoecia, a complete basalization occurs, and the majority of the gynoecium appears to be composed of style tissue. This is suggested by the appearance of style-like epidermal cells along the stalked and lower ovary regions of ett-1 gynoecia, and of style-specific reporter gene expression in the upper ovary part of ett-1 gynoecia. The latter represents both a basalized and an inverted development of tissues (see below). Inversion of the transmitting tract in ett gynoecia The inversion of identity in the inside-outside dimension is suggested by the expansion of functional transmitting tract in the abaxial direction (indicated by blue in Fig. 13). The similarities of the abaxial outgrowths and the normal transmitting tract include the spongy arrangement and staining of subepidermal cells with alcian blue and their ability to serve as functional transmitting tract for wild-type pollen. The inversion is also suggested by the appearance of ASA1:GUS expression in the external cell layers of ett surface outgrowths. ASA1:GUS expression in the wild-type style does not occur in the outer epidermis, and is restricted to the cells of and surrounding the transmitting tract and the postgenitally fused inner epidermis. Thus the ASA1:GUS expression in ett gynoecia suggests that the inverted transmitting tract tissue is more style-like than ovary-like, as the transmitting tract in the wild-type ovary does not exhibit ASA1:GUS activity. GUS expression in the novel unidentifiable cells of the outer epidermis of ett-3 and ett-1 gynoecia suggests that these cells could have an identity similar to the cells of the inner (normally fused) epidermis of the style. These novel cells also induce pollen germination. That decapitated styles will induce pollen to germinate (Kandasamy et al., 1994) supports the hypothesis that the novel outer epidermis cells in ett gynoecia are similar to wildtype inner epidermal style cells. Possible roles for ETT function These results suggest that ETT function is necessary for the coordination of positional information (Wolpert, 1969) along the longitudinal and transverse axes of the developing Arabidopsis gynoecium. It is clear that in the absence of complete ETT function, the correct development of cell types in their normal apical-basal and inside-outside order does not occur. The relationship between the resultant abnormalities of basalization 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 genetic deficiency results in both valve decrease and abaxial expansion of transmitting tract, or if it affects one of these processes directly, and the other secondarily. Likewise, it is unknown if the alterations in vascular patterning are causal or resultant of the alterations in apical-basal patterning. Nevertheless, some aspects of ett phenotypes suggest possible patterning events that occur during wild-type gynoecium development, as well as roles for ETT functioning in these events. It is known that ett gynoecia primordia are misshapen at the very earlieast stages of development (Alvarez, 1994; A. S. unpublished data). This suggests that ETT functions very early in the formation or elaboration of a prepattern specifying apical-basal and adaxial-abaxial patterning in the gynoecium primordium. The stochastic formation of valve tissue on the two lateral sides of an ett gynoecium suggests that the two sides develop independently, or that ETT function is necessary for their coordination. Lastly, the composition of stochastically forming sectors of valve tissue abutting style cells abutting stigmatic cells, suggests that a positional information mechanism exists for juxtaposing these cell types, and that ETT is involved in polarizing this information in one direction. Besides its role in global patterning, the data suggest that ETT function is necessary for the establishment of fields of valve tissue on the lateral sides of developing gynoecia. The most severely affected allele, ett-1, results in the complete absence of valve tissue. Double mutant analyses of ett-1 with homeotic mutations (ap2-2 and pi-1) that position carpel (valve) tissue in the first and third whorls of the flower indicate that valve tissue formation is only affected in the fourth whorl by ett-1(a.s. unpublished data). Thus, ETT appears to be involved in valve tissue formation only in the fourth whorl, and this function is not required for the differentiation of valve tissue per se. This suggests that ETT function is involved in the positioning of valve cell types rather than in their molecular differentiation. ETT participation in valve formation may be causative or restrictive. For example, ETT may act in the recruitment of cells to form a field of valve tissue. Alternatively, ETT may participate in positioning a style-valve boundary in the developing gynoecium, since this boundary is retained in ett-2 and ett-3 gynoecia, but shifted relative to the normal position. The mispositioning of this boundary could affect the potential size and position of a valve sector. In addition, ETT may be involved in establishing the polarity, or direction in which positional information is interpreted relative to boundary positions. This could explain the circular style-valve boundary in ett-3 gynoecia, as well as the reversal of polarity of the outside surface of ett gynoecia. The cloning of the ETT locus and the localization of its gene product during development might distinguish between these possibilities. Finally, some features of the ett phenotypes are not novel to botanists. The basal solid region of the internode-like structure found inbetween the ovary and the medial stamens of ett flowers is found in other members of the Brassicaceae where it is referred to as a stipe or gynophore. Likewise, the growth of stigmatic tissue at carpel margins (if the two carpel model is to be believed for Arabidopsis and the Brassicaceae) is not unheard of and may represent an ancestral condition of the angiosperms (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 gift of the ett-3 to ett-8 alleles, and to Kris Niyogi and G. Fink for the gift of the ASA1:GUS lines. We are also indebted to Judy Roe and Don Kaplan for help with all aspects of this work. We thank Tim Durfee, Sarah Hake, Fred Hempel, and Chad Nusbaum for critical comments on versions of this manuscript, members of the Zambryski lab for general assistance, Wilfred Bentham, Don Pardoe, and the Berkeley electron microscope facility for help with SEM, and Steve Ruzin and the NSF Center for Plant Developmental Biology for assistance with microscopy. A. S. thanks Miko Maruoka and Toshi Foster for encouragement and advice. This work was supported by Department of Energy grant 88ER13882 to P. C. Z. REFERENCES Alvarez, J. (1994). The ETTIN gene. In Arabidopsis: an Atlas of Morphology and Development (ed. J. Bowman), pp New York: Springer- Verlag. Alvarez, J. and Smyth, D. (1994a). The SPATULA gene. In Arabidopsis: an