We describe allelic series for three loci, mutations in which result in homeotic conversions in two adjacent whorls in the Arabidopsis thaliana flower. Both the structure of the mature flower and its development from the initial primordium are described by scanning electron microscopy. New mutations at the APETALA2 locus, ap2-2, ap2-8 and ap2-9, cause homeotic conversions in the outer two whorls: sepals to carpels (or leaves) and petals to stamens. Two new mutations of PISTILLATA, pi-2 and pi-3, cause second and third whorl organs to differentiate incorrectly. Homeotic conversions are petals to sepals and stamens to carpels, a pattern similar to that previously described for the apetala3-l mutation. The AGAMOUS mutations, ag-2 and ag-3, affect the third and fourth whorls and cause petals to develop instead of stamens and another flower to arise in place of the gynoecium. In addition to homeotic changes, mutations at the APETALA2, APETALAS and PISTILLATA loci may lead to reduced numbers of organs, or even their absence, in specific whorls. The bud and flower phenotypes of doubly and triply mutant strains, constructed with these and previously described alleles, are also described. Based on these results, a model is proposed that suggests that the products of these homeotic genes are each active in fields occupying two adjacent whorls, AP2 in the two outer whorls, PI and AP3 in whorls two and three, and AG in the two inner whorls. In combination, therefore, the gene products in these three concentric, overlapping fields specify the four types of organs in the wild-type flower. Further, the phenotypes of multiple mutant lines indicate that the wild-type products of the AGAMOUS and APETALA2 genes interact antagonistically. AP2 seems to keep the AG gene inactive in the two outer whorls while the converse is likely in the two inner whorls. This field model successfully predicts the phenotypes of all the singly, doubly and triply mutant flowers described.

Flowers of Arabidopsis thaliana originate as small outgrowths of cells on the flanks of the florally induced shoot apical meristem. These cells divide and differentiate, eventually producing a flower with a precisely defined pattern of four types of floral organs, with each type found in one of the four concentric whorls of the wild-type flower (Bowman et al. 1989; Smyth et al. 1990). During the developmental process, the cells in each flower primordium must in some way assess their positions, either globally or in reference to nearby cells, and they or their descendents must subsequently differentiate to the appropriate cell types. As an approach to finding the molecular mechanisms by which cells in developing flowers recognize and realize their fates, we study genes whose products are necessary for proper pattern formation in Arabidopsis flowers.

Several such genes have been described (Pruitt et al. 1987; Meyerowitz, 1987; Bowman et al. 1988, 1989; Komaki et al. 1988; Haughn and Somerville, 1988; Hill and Lord, 1989; Meyerowitz et al. 1989; Kunst et al. 1989; Yanofsky et al. 1990). The best-studied of them are a group of four genes whose mutant phenotypes include homeotic conversions of floral organs. These are the AGAMOUS (AG) gene, mutants of which have petals where stamens are found in wild type (the third whorl), and internal flowers in the place of the ovary (the fourth whorl in wild type); the APETALAS (AP2) gene, different mutant alleles of which cause different conversions in the outer two of the four whorls of the flower; and the APETALAS (AP3) and PISTILLATA (PI) genes. Mutations in the AP3 and PI genes cause sepals to develop in the positions occupied by petals in wild-type flowers (the second whorl), and have variable effects in the third (in wild type, stamen) whorl. To date, there are published descriptions of two mutant ag alleles, ag-1 (Pruitt et al. 1987; Bowman et al. 1988, 1989) and ag-2 (Yanofsky et al. 1990), seven mutant ap2 alleles that show a broad range of related phenotypes (ap2-1 through ap2-7-. Pruitt et al. 1987; Bowman et al. 1988, 1989; Komaki et al. 1988; Meyerowitz et al. 1989; Kunst et al. 1989), and one mutant allele each of ap3 (Bowman et al. 1989) and pi (Bowman et al. 1989; Hill and Lord, 1989).

Given the range of phenotypes seen in ap2 mutants, in which first whorl organs can be leaves or carpels, second whorl organs petaloid stamens, stamens, or absent, and third whorl organs normal or absent, it seemed worthwhile to extend the allelic series for each of the homeotic loci. In this paper, we report the phenotypes of one new ag allele, three alleles of ap2, each with phenotypes different from those described before; and two new alleles of pi. We also describe the mature phenotype and early development of a range of doubly and triply mutant combinations made with these new and previously described alleles.

These new data, along with those already published, have led us to a general and testable model of organ specification in Arabidopsis flowers. This model details the roles of the wild-type products of each of the homeotic genes in specifying organ identify in different regions of the developing flower. These data also provide new information on the roles of the wild-type products of the homeotic genes in establishing organ number and pattern in the flower.

The mutant alleles studied are listed in Table 1. All are recessive. The new mutations are in the Landsberg ecotype, homozygous for the erecta mutation, and were generated by mutagenesis of seeds with ethylmethane sulfonate (EMS), ag-1, ap2-l, pt-1, and ap3-l were obtained from Maarten Koomneef (Department of Genetics, Wageningen Agricultural University, The Netherlands) Wild-type alleles are symbolized in block capitals and italics; mutant alleles in lower case italics. Individual mutant alleles are designated by a number that follows the mutant symbol and a hyphen. Doubly and triply mutant strains were constructed by manual cross-polhnation, using as parents strains homozygous for individual mutations, except in the strains involving agamous alleles, which are sterile when homozygous, thus necessitating the use of heterozygotes as parents. The resulting F1 plants were allowed to self-pollinate, and double and triple mutants were selected from the F2 plants. Seeds were planted on a peat moss/potting soil/sand (3:3:1, v:v:v) mixture. The plants were grown in incubators under constant cool-white fluorescent light at 25 °C (unless otherwise stated) and 70% relative humidity.

For scanning electron microscopy (SEM), young primary inflorescences were fixed in 4% glutaraldehyde in 0.025 M sodium phosphate (pH 7 0) at 4 °C overnight, and then transferred to 1 % osmium tetroxide in the same buffer at 4°C overnight. They were rinsed in the same buffer and dehydrated in a graded ethanol series at 4 °C. This material was critical point dried in liquid carbon dioxide. Individual flowers were removed from inflorescences and mounted on SEM stubs. Organs were dissected from individual flowers using glass needles The mounted specimens were coated with gold and palladium (4:1) in a Technics Hummer V sputter coater following each dissection. SEM was performed on an ETEC Autoscan scanning electron microscope at an accelerating voltage of 20 kV, and the images recorded on Kodak 4127 or Polaroid 55 film

Wild type

Wild-type Arabidopsis thaliana flowers (Fig. 1A) contain four concentric regions (whorls), each occupied by a different organ type (Bowman et al. 1989). The first (outermost) whorl of the wild-type flower contains four sepals, two medial and two lateral (with respect to the stem of the inflorescence). The second whorl holds four petals, which are in alternate positions with the sepals. The third whorl includes six stamens, four long medial ones and two short lateral. The fourth whorl is occupied by the gynoecium, which consists of a two-chambered ovary topped with a short style, and capped with a stigma. Nectaries, which appear as small cellular mounds with stomata on top, are formed at the base of the stamens, though their presence is variable, so that in any flower some stamens may have them, and others may not. The individual cells that constitute each organ are characteristic of the organ type, so that both overall structure and cellular identity can be used as criteria for organ type.

The development of the flowers has been described in detail, and divided into twelve stages from the first appearance of a flower primordium, to anthesis (Müller, 1961; Bowman el al. 1989; Hill and Lord, 1989; Smyth et al. 1990; Fig. 2A). New flower primordia are added at the top of the inflorescence, where the growing point is, in a spiral phyllotactic pattern. The inflorescence is thus a raceme, and an individual inflorescence may contain a complete developmental series of flowers, from the youngest primordium at the apex, to mature fruits toward the base.

Single mutant strains

agamous

Two agamous alleles have been described previously, ag-1 (Bowman et al. 1989) and ag-2 (Fig. 1B, Yanofsky et al. 1990) and we report here the isolation of a third allele, ag-3 (Table 1) The ag-1 and ag-3 mutants have very similar phenotypes; the ag-2 mutant has a somewhat different appearance. In all the mutants, whorls one and two appear normal, while in whorl three the positions occupied by the six stamens in wild type are occupied by six petals, and in whorl four the cells where the gynoecium normally develops differentiate into a new flower, consisting generally of an outer whorl of sepals, and two whorls of petals. Since this process of formation of flowers within flowers continues, mutant flowers can consist of at least five nested flowers, with a total of 70 or more organs (Bowman et al. 1989). All of the mutants also show a less than perfect repetition in the inner flowers. Some inner flowers have fewer than 10 petals, and some of the organs may arise in ectopic positions. The internal sepalloid organs are often not perfect sepals, but can be mosaics of petal and sepal tissue, with the petal tissue always forming in longitudinal sectors on the outer margins of the organs. Development of ag-2 flowers resembles that of ag-1 flowers (Bowman et al. 1989), as can be seen in Fig. 2B. The one difference so far observed is that ag-2 homozygous flowers show a greater degree of pedicel elongation of the internal flowers than ag-1 and ag-3. Very likely this is due to a difference at the erecta locus, which when mutant reduces internode elongation throughout the plant. The ag-1 and ag-3 mutants are in the erecta background; ag-2 is not (Table 1). None of these ag alleles is temperature sensitive.

apetala2

Detailed descriptions are available for ap2-l (Bowman et al. 1989) and ap2-5, -6, and -7 (Kunst et al. 1989), and briefer descriptions of ap2-2 (Meyerowitz et al. 1989) and ap2-3 and -4 (Komaki et al. 1988) have also been published. We give here a detailed description of the flowers and of their development in homozygotes for three further alleles, ap2-2, ap2-8, and ap2-9 (Table 1). All of these show a greater departure from wild type than any of the mutants previously described. These descriptions extend the allelic series, and indicate new functions for the wild-type AP2 gene product. In addition, we found that the three alleles are all temperature-sensitive, as has been previously shown for ap2-l (Bowman et al. 1989), implying that the underlying process or the wild-type protein, rather than the mutant gene product specifically, is affected by temperature changes.

apetala2-2

The most extreme allele characterized to date is ap2-2. Flowers of plants homozygous for this allele consist mostly of carpelloid and staminoid organs (Fig. 1C, Tables 2-4). The identities of the outer two whorls of organs are altered and, in addition, the numbers and/or positions of organs in all four whorls may be altered. Phenotypic differences are observed between the lateral and medial first whorl organ positions and thus they are treated separately (Table 2). About half of the time, the lateral positions have no organ development. When organs do form they tend to be cauline leaf-like organs with stellate trichomes on their abaxial surfaces and stipules at their bases (both characteristics of leaf development) or carpelloid leaf-like organs with stigmatic tissue at their tips and rudimentary ovules along their margins (Table 2). Each of these organs may have the long abaxial epidermal cells characteristic of sepals. The lateral first whorl positions can also be occupied by filamentous structures, some of which have leaf-hke character (such as stellate trichomes) while others are Brassicaceae (Arber, 1931) and in apl-1 flowers of squamule-like structures, thin filamentous outgrowths Arabidopsis (Irish and Sussex, 1990; D.R.S. and of cells that have been observed in other species of J.L.B.). In contrast, the medial first whorl positions are always occupied and exhibit a greater degree of carpellody. Solitary carpels capped with stigmatic tissue, and exhibiting marginal ovules are the most common organ in these positions (Table 2). Scanning electron microscope analysis of the epidermal cells of these organs shows cellular morphologies ranging from carpel-like (with a lower ovary-like portion of regular cellular files, and an upper sty lar portion) to cauline leaf-like (consisting of irregularly shaped cells with occasional stellate trichomes). On occasions, these carpelloid medial first whorl organs are fused to the central, fourth whorl gynoecium. In many cases, the medial first whorl organs are mixtures of stamen and carpel tissue (Table 2, Figs 1C, 2C). The staminal portion is always positioned at the outer margins of the organs, while the carpelloid tissue is central. The stamen sectors have both epidermal and internal cellular morphologies resembling those of wild-type stamens, but the central carpelloid sectors have cellular morphologies ranging from those characteristic of carpels to leaf-like. Individual, phenotypically mosaic organs of this type can contain both ovules and pollen.

There are no organs in the positions occupied by second whorl organs in wild-type flowers in ap2-2 homozygotes (Table 3). This is due to a failure to form organ primordia. The third and fourth whorls are affected with respect to position and number but their identities are not altered. Third whorl positions are largely unoccupied (Table 4, Fig. 1C). When the third whorl organs are present, they are normal and fertile stamens and tend to occupy the lateral positions. Two carpels occupy the fourth whorl but they often fail to fuse properly (Table 4). In addition, when they do fuse correctly, the resulting gynoecia are usually not oriented as they would be in wild type (Fig. 2C). In 14 out of 15 fused carpel gynoecia of ap2-2 homozygotes scored, they were either twisted, or oriented 90 ° from the wild-type orientation. The gynoecium is nonetheless functional, setting seed when manually pollinated.

The development of ap2-2 flowers diverges morphologically from that of wild type as early as stage three, the stage when the organ primordia of the first whorl arise (Fig. 2C). This is slightly after the beginning of the temperature-sensitive period (stage two) that was determined for second whorl organ development in ap2-l homozygotes (Bowman et al. 1989). The medial first whorl organs are enlarged relative to wild type, and grow rapidly, with the cell files typical of gynoecial development. These organs develop ovules on their margins and stigmatic papillae at their apices prior to the formation of the same features in the central gynoecium. Stipules, which are characteristic of early leaf development (Smyth et al. 1990), and which are seen at the base of developing first whorl organs of ap2-1 homozygotes (Bowman et al. 1989), are not seen in medial first whorl ap2-2 organs. Lateral first whorl primordia, when present, appear to originate lower on the pedicel of the flower primordium than organs in the same position in wild-type flowers (Fig. 2C) and the organs that develop in these positions often have stipules at their bases. The occasional stamens that are observed developing in third whorl positions occupy the lateral positions, and there are a maximum of two stamens in any developing flower. In wild type, nectaries are always associated with stamens, while in ap2-2 homozygotes nectaries can be present regardless of the occurrence of stamens. The remaining floral meristem gives rise to the gynoecial cylinder. Its growth is often irregular, and the division between the two carpels occurs in an abnormal orientation. Observations on developing mutant flowers show that the absence of organs in positions normally occupied in the second and third whorls does not result from failure of initiated organ primordia to develop, but rather results from the failure of primordia to appear in these positions. This also occurs in most of the lateral first whorl positions that lack organs; occasionally, however, a small mound of cells may occur in these positions in which no organ develops. It is unclear whether these outgrowths represent aborted organ primordia or some other structure. Thus, in addition to changes in organ identity, mutations in the ap2 locus change basic organ number in flowers.

When ap2-2 plants are grown at low temperature (16 °C), the frequency of occurrence of lateral first whorl and all third whorl organs is increased compared to 25 °C. Nonetheless, second whorl organs fail to develop. In addition, the first whorl organs may fuse to each other as described below for ap2-9 flowers grown at 16 °C. When they are grown at 29 °C, ap2-2 homozygotes frequently fail to produce any first, second or third whorl primordia at all. This results in flowers that contain only a gynoecium, which is typically composed of two carpels oriented at 90 ° from the positions occupied by the two carpels in wild type.

apetala2-8

Flowers of plants homozygous for the ap2-8 allele are similar to those of ap2-2 homozygotes in both their mature phenotype (Tables 2 –4) and their ontogeny (Fig. 2D). The ap2-8 homozygotes are also similar to those of ap2-2 in their response to different growth temperatures, with greater organ loss and more complete organ conversions at higher temperatures, and less departure from wild type at lower temperatures (Fig. 3).

apetala2-9

Plants homozygous for the ap2-9 mutation have flowers with a phenotype slightly closer to wild type than plants homozygous for ap2-2 or ap2-8 (Fig. ID). Briefly, the medial first whorl organs are carpelloid and the lateral first whorl organs often fail to develop (Table 2). No organs form in the second whorl positions (Table 3) and the third whorl is also largely unoccupied (Table 4). When third whorl organs are present, they form normal fertile stamens. Two carpels occupy the fourth whorl, but they often fail to be properly fused (Table 4).

When grown at 16 °C, flowers homozygous for ap2-9 exhibit both numerical and positional abnormalities (Fig. 2E). Although fewer organs are missing than in flowers grown at higher temperatures, those organs that are present tend to occupy ectopic positions and are often fused to each other. For example, lateral first whorl primordia are frequently present, though they appear to arise lower on the pedicel of the developing flower than do lateral first whorl organ primordia in wild type. These organ primordia differentiate to become carpelloid leaves, which are often fused to the more-carpelloid medial first whorl organs to form a connate ring, with marginal ovules and stigmatic tissue at its top (Fig. 2E). Sometimes a fifth organ primor-dium arises between the positions ordinarily occupied by first and second whorl organs (Fig. 2E). Second whorl organs appear to be absent, though there are stamens, carpelloid stamens and solitary carpels whose positions do not allow their assignment to the second or third whorl. Third whorl organs, which also arise in ectopic positions, are normal stamens, and the ovary is usually normal.

trans-hetero zygotes

In addition to characterizing the phenotype of homozygotes of each of the three extreme ap2 alleles, we have made all possible heterozygous combinations of these alleles with each other, and with ap2-1, the allele whose horpozygotes show the least departure from wild type (at 25 °C, four first whorl leaves, four second whorl petalloid stamens, normal third and fourth whorls). In each case, the phenotype of the heterozygotes is intermediate between that of the homozygotes for each allele involved. ap2-1/ap2-2 heterozygotes (Fig. 2F, Tables 2 –4), representing a combination of the two extremes of the ap2 allelic series, are discussed because they emphasize aspects of the ap2 phenotype, in particular the difference in phenotype between the lateral and medial first whorl organs, the failure of organ primordium formation in the second and third whorls, and the abnormal positions of the remaining second and third whorl organ primordia. ap2-l/ap2-2 flowers have lateral first whorl organs that resemble cauline leaves, usually (76 % of 72 organs scored) with stigmatic tissue at their tips, whereas the medial first whorl organs are much more carpelloid (Table 2, Fig. 2F). These positions are occupied by solitary carpels or carpelloid leaves, with ovules at their margins in addition to stigmatic papillae at their tips. The carpellody of the first whorl organs increases with the age of the inflorescence, with the cauline leaf-like organs present only on the first few flowers, and the solitary carpels generally present in later flowers. This trend has been observed for all the ap2 alleles. Second whorl organs are usually absent and their absence is due to the failure of organ primordia to form, not to failure of initiated primordia to develop (Fig. 2F). When they are present, the second whorl organs range in phenotype from morphologically normal stamens to staminoid petals. Third whorl organs are normal, and occupy the same positions as in wild type when all six are present. When fewer than six are formed some may be in ectopic positions. The nectaries are normal, as is the gynoecium. The phenotype of ap2-1/ap2-9 heterozygotes (Tables 2-4) is slightly closer to wild type than that of ap2-1/ap2-2 heterozygotes. Fig. 3 illustrates the ap2 allelic series, as a summary of the very broad range of phenotypes that result from the different mutant alleles at this locus.

pistillata and apetala3

Mutants homozygous for one allele each of these two unfinked loci have been described before (Bowman et al. 1989; Hill and Lord, 1989). Homozygotes for pi-1 have a normal first whorl of four sepals but the organs of the second whorl develop as sepals rather than petals. The cells that would in wild type form the organs of the third whorl appear to be largely incorporated into the gynoecium, which is, as a consequence, abnormal in size and irregular in structure. The gynoecium usually has more than two carpels and often there are thin, filamentous structures partly or fully fused to its sides (Bowman et al. 1989; Hill and Lord, 1989). Homozygotes for ap3-l, when raised at 25 °C, have the same second whorl abnormality seen in pi-1, but the six third whorl organs are usually carpels or staminoid carpels, either solitary or connate with other members of the same whorl (Table 5). At 29 °C the third whorl organs in ap3-l flowers can also be free filamentous structures which are thinner than the filaments associated with wild-type stamens but usually thicker than the filamentous structures of pi-1 flowers (see Fig. 7D of Bowman et al. 1989). The two new alleles of pistillata, pi-2 and pi-3 (Table 1) have phenotypes intermediate between those of ap3-l and pi-1, with the allelic senes (in order of mcreasing departure from wild-type organ number and gynoecial morphology) pi-3, then pi-2, then pi-1. None of these pi alleles is temperature sensitive.

pi-2

Flowers homozygous for pi-2 (Fig. 2G) have a normal first whorl of sepals, and like pi-1 homozygotes, have four additional sepals in the positions normally occupied by petals in wild type. The six third whorl organ positions may be occupied by filamentous structures, by carpelloid organs, or organs may be absent (Table 5). The filamentous structures, which may be capped with stigmatic papillae, are thinner than the filaments associated with wild-type stamens but usually thicker than those observed in pi-1 flowers. The third whorl organs present are often congenitally fused to the central gynoecium, producing an irregular gynoecial morphology (Fig. 2G, second and third panels). In other cases, the gynoecium is normal.

pi-3

Homozygotes for pi-3 are very similar to ap3-l mutants. They have second whorl sepals and, in the third whorl positions, either solitary carpels, staminoid carpels, or filamentous structures such as those described for pi-2 flowers (Table 5). The lateral third whorl organs are more staminoid than those occupying medial positions, and the extent of carpellody in all third whorl positions increases with the age of the inflorescence (as in ap3-l).

pi-1/pi-2

Heterozygotes of genotype pi-l/pi-2 (Fig. IE) are intermediate between the two homozygous types in third whorl organ identity. Many (33 % ) of these organs are fused to the central gynoecium, regardless of their identity A summary of the changes in organ identity in the third whorl, with increasing departure of the pi phenotype from normal, is staminoid carpel, carpel, filamentous structure; with fusion to the fourth whorl organs also increasing with increasing departure from wild type (Table 5).

In all pi mutants, the nectaries are normal and occupy their wild-type positions (see Smyth et al. 1990). When (as in pi-1 and some pi-2 positions) there is no third whorl organ, the nectaries are still present between the sepals occupying the second whorl and the gynoecium. Tire presence of nectaries thus does not depend on the development of stamens or any third whorl organ. In all pi mutants, and in ap3-1 mutants, the sepals in the second whorl are smaller than the sepals occupying the first whorl, and they develop on a relatively delayed time course, as do petals relative to sepals in wild-type powers (Bowman et al 1989; Hill and Lord, 1989).

Doubly mutant strains

Many of our current conclusions about the domains of action of the various homeotic gene products, and about their interactions, come from study of the phenotypes of strains homozygous for different pairs of the homeotic mutations. We have previously described the phenotypes of the doubly mutant strains ag-1 pi-1 ; ag-1 ap3-l\ ag-1 ap2-1;, ap2-l ap3-1; and ap2-1 pi-1 (Bowman et al. 1989). The alleles of ag and pi used in these studies display the most extreme phenotypes of those so far observed m their respective allelic series; they may thus represent null or close to null phenotypes. In contrast, the allele of ap2 used, ap2-1, is the weakest allele in the series of mutations known at this locus. Consequently, we have constructed more doubly mutant strains, using the allele at the ap2 locus that shows the greatest departure from wild type, ap2-2. We have also constructed an ap2-1 pi-2 strain, to observe the effect of using a weaker pi allele in one combination. In addition, we present here new information on flower development in the doubly mutant strains whose mature phenotypes were previously described (Bowman et al. 1989).

ap2-l pi-1, ap2-l pi-2, and ap2-l ap3-l

Flowers homozygous both for ap2-1 and pi-1 have a first whorl of four slightly carpelloid leaves (as observed in ap2-l alone, but with a slightly increased degree of carpellody). The second whorl contains 1 to 4 organs that range from cauline leaf to solitary carpel, with most mixtures of the cell types characteristic of these organs, and none showing staminody. Third whorl organs are absent, as in pt-1 alone, and the gynoecium resembles that of pi-1 flowers (Bowman et al. 1989). When a weaker pi mutant is used, such as in ap2-l pi-2, flowers exhibit a phenotype more similar to that of ap2-l ap3-l flowers (Bowman et al. 1989) than to that of ap2-1 pi-1. The outer whorl is occupied by cauline leaf-like organs that can have stigmatic tissue at their tips. Second whorl organs range in phenotype from cauline leaf-like organs to organs that have characteristics of leaves, stamens and carpels. The third whorl organs are solitary carpels or filamentous structures such as those described for the third whorl of pi-2 flowers.

In each of the genotypes, both the second and third whorl organs are frequently absent. When all the positions in a whorl are occupied by an organ, the organs occupy the correct positions. When one or more are missing, however, the remaining ones develop from organ primordia that are often enlarged and occur in ectopic positions (Fig. 4A,B). In contrast, when second whorl organs are absent from ap2-l flowers, the positions of the remaining organs are not altered and second whorl organs are never absent in ap3-l, pi-1, or pi-2 flowers. The development of the gynoecial cylinder is often abnormal, resulting in an unfused ovary that may also be adnate with both second and third whorl organs. The fusion between second and third whorl organs and the central gynoecium is congenital. Observations on developing flowers show that missing organs result from failure of organ primordium formation (Fig. 4A,B).

ap2-2 pi-1

When a plant is homozygous for both the strong alleles, ap2-2 and pi-1, many organs are absent (Fig. 5A). The lateral first whorl positions may be occupied by cauline leaf-like organs (11 out of 66 positions scored), carpelloid leaves, (9/66), or filamentous structures (22/66). In 24 of 66 positions, no organ was observed. The filamentous structures observed are either filamentous leaf-like structures or thinner squamule-like structures, such as those described in the lateral first whorl positions of ap2-2 flowers. An example of a squamule-like structure is shown in the fourth panel of Fig. 4C. The remainder of the ap2-2 pi-1 flower consists only of a central gynoecium, which usually consists of four carpels (30 out of 33 flowers analyzed; Fig. 4C). In the first five or so flowers produced on any inflorescence, the carpels in the medial positions have stellate trichomes on their abaxial surfaces, while the lateral carpels are bare (Fig. 5A). Small domes of cells may be present between the positions of the lateral outer whorl organs (or the positions where they would be expected to be, were they present) and the central ovary. These are apparently nectaries, since they anse late in development, when the gynoecium develops its stigmatic papillae, and since stomata occur at the dome apices (Fig. 4C).

Observations on the development of ap2-2 pi-1 flowers show that the two medial first whorl organ primordia do form, and are enlarged relative to the size of wild-type medial first whorl primordia. These primordia are fused to each other as they develop, forming a cylinder that will later be a part of the central gynoecium (Fig. 4C). The cells central to these primordia, which in wild type would develop into the second, third and fourth whorls, appear to be recruited into the growing cylinder formed by the fused medial first whorl primordia. Thus, the mature gynoecium appears to consist of two medial (and slightly phylloid) carpels that originate in the first whorl, and two lateral fourth-whorl carpels. The absence of any organs developing from the second and third whorls is consistent with the phenotypes of the single mutant homozygotes, since ap2-2 mutants lack second whorl organs, and pi-1 mutants lack those of the third whorl. As seen in ap2-2 flowers, the lateral first whorl organs that form develop from primordia produced lower on the pedicel of the flower primordium than do first whorl organ primordia in wildtype flowers.

ap2-2 ap3-l

Homozygotes for the strong ap2-2 allele and ap3-1 produce flowers that consist primarily of carpelloid organs, with occasional cauline leaf-like organs in the lateral first whorl positions. All of the carpelloid organs may be fused, such that the flower resembles that of ap2-2 pi-1 homozygotes, or the medial first whorl organs may be separate from the central gynoecium. Organs do sometimes form between the organs of the first and fourth whorls. These may be third whorl organs, as sometimes form in ap2-2, but they are carpels and not stamens (as in ap2-2) as a result of the absence of the wild-type action of the AP3 gene.

ap2-l ag-1

Our earlier observations of ap2-l ag-1 double homozygotes showed that the flowers retain the indeterminate growth characteristic of ag-1 homozygotes, but have additional changes in organ identity (Bowman et al. 1989). The first floral whorl contains four cauline leaves with stellate trichomes and stipules, as well as an epidermal cellular morphology typical of leaves (Fig. 4D). These organs have stigmatic tissue at their tips very infrequently in contrast to the first whorl organs of ap2-1 flowers that are often capped with stigmatic papillae. The second and third whorl organs are intermediate between petals and stamens, both at the organ level, and in the appearance of individual epidermal cells. Observations of the development of these flowers shows that all organs of the first three whorls originate from primordia that are in the positions and numbers of the similar primordia in wild type (Fig. 4D). The cells that would ordinarily develop into the gynoecium form, as in ag-1 homozygotes, an inner flower, whose organ number and positions may differ from those typical of wild-type flowers.

ap2-2 ag-1

Remarkably, ap2-2 ag-1 double homozygotes (Fig. 5B) strongly resemble ap2-l ag-1 double homozygotes: the absence of wild-type AG activity eliminates many of the phenotypic differences between flowers homozygous for the different ap2 alleles. This strongly implies that these differences are due to different interactions of the products of the ap2 alleles with the wild-type AG gene or its product, a model that will be developed in the Discussion.

The phenotype of the ap2-2 ag-1 double homozygotes includes lateral first whorl organs (which were present in 32 out of 44 positions counted) resembling either cauline leaves (22/32 organs) or filamentous structures (10/32 organs). The filamentous structures may be either leaf-like or squamule-like structures, such as those described for lateral first whorl positions of ap2-2 flowers. Six of the 22 phylloid organs had stigmatic tissue at their apices. The medial first whorl organs are carpelloid leaves (in contrast to the solitary carpels, with few leaf-like characteristics, found in the ap2-2 single mutant). These carpelloid leaves have an epidermal cellular morphology of leaves, often develop with stipules at their bases, and frequently have stellate trichomes on their abaxial surfaces (Fig. 5B). Long epidermal cells of the type found only on the abaxial surface of sepals also are present, however, and stigmatic papillae and ovules may develop on the tips and margins, respectively.

Second whorl organs do occur in the double mutant, again in contrast to ap2-2 single homozygotes. Those second and third whorl organs that form are indistinguishable from each other and have both an overall morphology and individual epidermal morphologies intermediate between those ordinarily seen in petals and stamens (Fig. 4E). The organs have rudimentary locules in which pollen grains are produced, but they do not dehisce. It is of note that the number of second and third whorl organs that develop (7.6 on average) is closer to the wild-type number (10) than the number of these organs found in flowers homozygous for ap2-2 alone (average 0.25). The second and third whorl organs in the double homozygote arise in roughly the normal pattern (Fig. 4E).

The remaining floral meristem, which in wild type would give rise to the gynoecium, develops into another flower primordium, as in ag-1 flowers. The organ primordia in this (and subsequent) inner flowers differentiate in the same patterns as those of the outermost flower, with a whorl of carpelloid leaves surrounding two whorls of stamen-petal intermediate organs. Organs that resemble carpelloid leaves (in whorls 1, 4, 7, etc.) may have longitudinal stamen-petal hybrid sectors along their margins, similar in pattern to the hybrid organs observed in the analogous positions in ag-1 flowers.

The ag-1 mutation thus suppresses both the extreme carpellody observed in the first whorl organs of ap2-2 flowers, and also suppresses the loss of second and third whorl organs characteristic of ap2-2 flowers. The fact that the ag-1 mutation exhibits phenotypic effects in the first and second whorls in a background homozygous for ap2-2, but shows no effects in these whorls when alone, suggests that the AG and AP2 genes interact at some level.

ag-1 pi-1 and ag-1 ap3-l

Finally, we will consider two other doubly mutant combinations involving agamous. Both ag-1 pi-1 and ag-1 ap3-1 flowers consist of an indeterminate number of whorls of sepals (Bowman et al. 1989; Fig. 5C,D). The developmental basis of the phenotypes in the two different genotypes is somewhat different; ag-1 pi-1 homozygotes consist of a repeating pattern of two whorls, whereas ag-1 ap3-1 flowers have a repeating pattern of three whorls. An ag-1 pi-1 double homozygote is shown in Fig. 6A. The four outer whorl and four second whorl organ pnmordia are initiated in the correct positions and all of these primordia develop into sepals. The four second whorl primordia are small and develop much more slowly than the outer whorl sepals, with the result that the second whorl of sepals remains smaller than the outer whorl sepals. The cells that would normally give rise to the third and fourth whorl organ primordia behave as if they were another flower primordium, developing four organ primordia on the margins of the remaining dome of meristematic cells. This process repeats indeterminately, as seen in ag-1 flowers, thus producing a flower with many whorls, all of which have four sepals, ag-1 ap3-1 flowers also consist of an indeterminate number of sepals (Fig. 5D but, in contrast to ag-1 pi-1 flowers, the pattern of organ pnmordia formation is indistinguishable from that observed in ag-1 (Fig. 6B), thus consisting of repeats of three whorls, with four organs in each of the outer two whorls, then six organs in the third. Each of the organs in both ag-1 pi-1 and ag-1 ap3-l flowers consist of cell types characteristic of wild-type sepals, complete with long epidermal cells on their abaxial surfaces.

Triply mutant strains

To extend our genetic analysis of the Arabidopsis homeotic genes, we have constructed four different strains, each homozygous for mutant alleles of three of the four homeotic loci. We will first discuss three of these strains together, since their phenotypes are similar. These strains involve ap2-1 and ag-1 in combination with ap3-l, pi-1, or pi-2.

ap2-l ag-1 pi-1, ap2-l ag-1 ap3-l, and ap2-l ag-1 pi-2

Each of these strains produce flowers that exhibit the indeterminate growth caused by the ag mutation and, in addition, all of the organs of the flower resemble cauline leaves (Fig. 7A,B). The organs are cauline leaflike in several aspects: they have stellate trichomes on their abaxial and, less frequently, on their adaxial surfaces, stipules are present at the base of most of the organs (Fig. 8A,B), they have an epidermal cellular morphology characteristic of leaves, and they senesce on the time course characteristic of leaves, not that typical of sepals (which begin to senesce soon after anthesis). They do, however, have the long epidermal cells on their abaxial surface that are characteristic of wild-type sepals.

Occasionally (in about 5 % of the flowers) in each triply mutant genotype a secondary flower is formed in the axil of a lateral first whorl organ. These flowers have their own pedicel and the same phenotype as the flower in which they arise. This phenomena has also been observed in ap2-1, ap2-1 ap3-1, ap2-1 ag-1, and ap2-1 pi-1 flowers at a lower frequency, and occurs in almost every flower with the apl-1 genotype (McKelvie, 1962; Irish and Sussex, 1990).

In ap2-l ag-1 ap3-1 flowers, outer whorl organ primordia are initiated in correct positions and the second whorl organ primordia are usually in wild-type positions and numbers (Fig. 8A). By contrast, the third whorl organ primordia are variable in number, position and size (Fig. 8A). The remaining floral meristem continues to produce organ primordia but in an ill-defined pattern. All of these organ primordia develop into cauline leaf-like organs (Fig. 7A).

In ap2-1 ag-1 pi-1 flowers, the identity of each organ is also that of leaf (Fig. 7B), but the pattern of the organs is different from that of ap2-l ag-1 ap3-1. The pattern in which the organs appear deviates from that of wild type at stage 5, when the second and third whorl primordia are normally formed (Fig. 8B). The pattern initially resembles that seen in ag-1 pi-1 flowers. The four first whorl primordia are initiated in the correct positions, as are the second whorl organ primordia in most cases. The remaining floral meristem then behaves as if it were a new flower, producing four more primordia on the margins of the remaining dome of cells. At this point, the floral meristem continues to produce organ primordia, but in no easily defined pattern.

Flowers of genotype ap2-l ag-1 pi-2 have been analyzed at the resolution of light microscopy. At this level, they appear similar to the flowers of the ap2-l ag-1 ap3-l strain.

ap2-2 ag-1 pi-1

The final genotype constructed, ap2-2 ag-1 pi-1, involved the strongest mutant alleles thus far isolated. Flowers on these triple homozygotes are composed of an indeterminate number of slightly carpelloid leaf-like organs (Fig. 7C) similar to those observed in the medial first whorl positions of ap2-2 ag-1 flowers. Each of the organs has an overall morphology intermediate between cauline leaves and carpels. The epidermal cellular morphology is characterized by irregularly shaped cells interspersed frequently with stomata, which is a phylloid appearance, though the abaxial surface also has many of the very long cells that are normally characteristic of sepals. Stellate trichomes, again a leaf character, may form on the abaxial surfaces, with their frequency decreasing on the inner organs. The most cauline leaf-like organs, which usually develop in what appears to be the lateral first whorl positions, sometimes have stipules at their bases. Stigmatic papillae develop at the tips of most organs, and ovules may develop along the margins of the organs from what resembles placental tissue.

Development is altered as early as stage 3, when the first whorl organ primordia appear (Fig. 8C). In ap2-2 ag-1 pi-1 flowers, the medial first whorl organ primordia appear in the correct positions, but their growth is abnormal, and they differentiate into carpelloid leaves. The lateral first whorl primordia sometimes fail to form (they are present only in 11/18 positions analyzed) and when they do form they appear to arise lower on the flower primordium than the position of the analogous organs in wild-type flowers. These lateral primordia differentiate either into cauline leaf-like organs with few carpelloid features (7/11 organs), or into filamentous structures (4/11 organs) like those described for lateral first whorl positions of ap2-2 flowers. The remaining floral meristem produces two more organ primordia in lateral positions, directly inside the lateral first whorl organs when these are present, and interior to all of the first whorl organs (Fig. 8C). This is followed by the production of two additional primordia in medial positions, interior to all of the previously produced primordia. This pattern of pairs of lateral, then medial organ primordium formation repeats indefinitely, and eventually each of the primordia develops into a carpelloid leaf. Occasionally, the medial first whorl organs and the next two inner organs are congenitally fused, forming a ring of organs. The lateral first whorl organs have not been observed to fuse with any other organs.

The phenotypes of each of the singly, doubly and triply mutant flowers are summarized in Table 6.

Each of the genes, AG, AP2, AP3, and PI, seems to act by allowing cells to determine their positions within the developing flower, and thus to act in allowing the cells to differentiate (or to direct their progeny to differentiate) appropriately. In addition, the AP2, AG and PI products appear to be involved in the initial production of floral organ pnmordia, which precedes their differentiation. Mutations in these genes thus result in both the misinterpretation of positional information, causing organ primordia to differentiate improperly, and the disruption of floral organ number and pattern. We have derived a model, presented below, which explains how these four genes could specify the identity of the floral organs. The model has been successful in predicting the phenotypes of doubly and triply mutant flowers, and in predicting the pattern of expression of one homeotic gene that we have cloned and analyzed so far (AG, Yanofsky et al. 1990). The model does not, however, address the issue of how these and other genes act to specify the position and number of organs in the developing flower. The model is also insufficient to explain all of the organ identity phenotypes seen. It is clear, then, that what follows is an overly simple, first- order model, which will require considerable modification as new mutations, and new phenomena, are described.

A simple model

The model is depicted in Fig. 9. The Arabidopsis wildtype flower consists of four concentric whorls of organs, arranged in a stereotypic pattern in terms of number and position. Each of the four mutations affects the differentiation of two adjacent whorls of organs, and thus falls into one of three classes: those affecting the first and second whorls, those affecting the second and third whorls, and those affecting the third and fourth whorls. The flower primordium can, consequently, be divided into concentric fields made up of pairs of adjacent whorls. The term ‘field’ is introduced here to refer to pairs of adjacent whorls, since these pairs of whorls, rather than single whorls, appear to be the domains of action of each of the classes of homeotic genes described. Field A is made up of whorls 1 and 2, and is the domain of action of AP2, the product of the AP2 gene. Whorls 2 and 3 constitute field B, which is the domain of AP3 and PI; and field C is made of whorls 3 and 4, the domain of AG action. AP3 and PI are grouped together for the remaining discussion because a mutation in either one results in a similar phenotype with respect to organ differentiation, and because the double mutant phenotype is indistinguishable from the single mutant phenotypes (Bowman et al. 1989). In what follows, it is important to recognize that by ‘whorl’ we mean a geographic location, within which organs of any identity can arise, and not the group of organs themselves. Whorls are thus identified by their position in the developing flower, and by the number and disposition of the organs within them, but not by the identity of these organs (see Bowman et al. 1989 for a longer discussion of this point).

Underlying our model is the assumption that the combination of homeotic gene products present in each whorl is responsible for specifying the developmental fates of the organ primordia that form in that whorl. The three overlapping fields of gene action thus specify each of the four different whorls of organs in the flower: in any whorl in which AP2 alone is active, sepals form. If both AP3 and PI are also present in combination with AP2, as in the wild-type second whorl, any organ primordia that are present differentiate into petals. Similarly, if AG alone is present, it directs any primordia in that whorl to develop into carpels, as is the case for the wild-type fourth whorl. If AP3 and PI are present in combination with AG, stamens are specified, as in the wild-type third whorl.

The final aspect of this simple model is that the AP2 and AG products are mutually antagonistic, such that AG prevents the action of AP2 in the third and fourth whorls of a wild-type flower, and AP2 prevents the action of AG in the first and second whorls. This proposal is based on observations that the ag-1 mutation has phenotypic effects m the first and second whorls in an ap2-2 mutant background, while no first or second whorl effects are seen in ag-1 singly-mutant flowers. In ap2-2 ag flowers the first whorl develops as carpelloid leaves rather than carpels as in ap2-2 flowers, and second whorl organs, which are missing in ap2-2 flowers, are partially restored in the double mutant and have a phenotype different from that observed in any ap2 allele alone. Likewise, the ap2 mutations have third and fourth whorl homeotic effects when combined with ag-1, but not when alone; ap2-2 ag-1 flowers have third whorl organs intermediate between petals and stamens, while ag-1 alone has normal petals in the third whorl. If the first whorl of the inner flower in an ag mutant is considered equivalent to a wild-type fourth whorl, ap2-2 also has a fourth whorl effect in an ag background, namely, conversion of these fourth whorl sepals into leaves or carpelloid leaves. According to this part of the model, then, if the AP2 product is missing, the phenotypic expression of AG expands into field A (whorls 1 and 2) and conversely, if the AG product is absent, the phenotypic expression of AP2 expands to include field C (whorls 3 and 4).

If one or more of the homeotic gene products is missing, the distribution of remaining products determines organ identity. For example, if either the AP3 or PI gene product is missing, leaving only those products specifying fields A and C, then the outer two whorls should develop into sepals and the inner two whorls into carpels. This is indeed the case in ap3-l, and pi-3. If the AP2 product is missing, AG is active in both fields A and C. The first and fourth whorl organs should then be carpels, and the second and third whorl organs, which develop in the presence of AG and AP3/PI, should be stamens. This is observed in the ap2 allelic senes; all those organs arising from cells that would normally give rise to second and third whorl organs are staminoid, whereas those arising from cells normally forming first and fourth whorl organs are carpelloid. Conversely, if the AG product is missing, the AP2 product should be active in both fields A and C (with, as in wild-type, AP3/PI active in field B), and a flower in which the first and fourth whorl organs are sepals, and the second and third whorl organs are petals should (and does) develop.

Predictions for double and triple mutants

The model makes clear predictions for the phenotypes of doubly and triply mutant flowers. For example, in ap3 ap2 and pi ap2 flowers, the AG product (and none of the others) should be present in all four whorls, since the AP2 product is not present to repress the activity of AG in the outer two whorls. Therefore, the flowers are expected to consist entirely of carpels. This is the case in such doubly mutant flowers (Figs 4C, 5A). A similar argument can be made to predict flowers composed entirely of sepals for ag pi and ag ap3 double mutants, and this prediction is confirmed by the phenotypes of these flowers (Figs 5C,D; Bowman et al. 1989).

The prediction for ag ap2 flowers is more difficult to make, because if both the AG and AP2 products are absent, only the AP3/PI products remain in field B. This is a distribution of homeotic gene products that does not match that thought to be found in any whorl of wild-type flowers. In wild type, the formation of both petals and stamens is dependent on the AP3/PI products. The decision as to whether to produce either a petal or a stamen, however, is dependent on the presence of either the AP2 product or the AG product, respectively. Thus the second and third whorl organs might be expected to have characteristics of both petals and stamens, but to be neither wild-type petals nor wild-type stamens. This is what is observed in ap2-2 ag-1 flowers (Fig. 4E). The phenotype of first and fourth whorl organs in the ap2-2 ag double mutants is also difficult to predict, since whorls 1 and 4 are predicted to have none of the homeotic gene products present in them, a situation with no precedent in the wild-type flower. Thus, the organs occupying these positions should not be of a type normally found in flowers. Indeed, the organs of these whorls in ap2-2 ag-1 flowers have characteristics of leaves (Fig. 5B).

It is tempting to speculate that the known three homeotic pathways are sufficient to specify floral organs and that, in their absence, only vegetative organs can form. The developmental ground state, however, appears not to be entirely vegetative, since the organs in the first and fourth whorls of ap2-2 ag-1 flowers are carpelloid leaves. If these organs indeed exhibit the developmental ground state, all floral organs should be carpelloid leaves in the triple mutant, in which all three of the homeotic pathways are blocked. This prediction is also confirmed; all organs in ap2-2 ag-1 pi-1 are carpelloid leaves (Figs 7C, 8C).

Each of these genes appears to control the specification of organ identity by regulating genes that are involved in the production of cellular and morphological characteristics of the different organ types, and not by directly specifying the final differentiation products. This is clear from the fact that organ types removed by single mutations may be partly restored by additional mutations. For example, ag mutants have no stamens, but in ap2 ag doubly mutant strains, the second and third whorl organs are in many respects staminoid. Another example is that single ag mutants have no carpelloid structures, but such structures are found in the ap2-2 ag-1 pi-1 triply mutant strain.

ap2-l: a special case

The phenotypes of ap2-l flowers seem to refute the organ specification model: the first whorl of ap2-l flowers is composed of leaves with few carpel characteristics, not the carpels predicted by the model. This apparent exception could be explained by proposing that in the ap2-1 allele, only one of the two proposed functions of AP2 has been significantly affected. According to our model, AP2 has two functions: one is to prevent AG from acting in the outer two whorls (field A), and the other is to affect directly the expression of the genes that lead to the differentiated phenotypes of sepals and petals. If the ap2-1 gene product fails to activate downstream genes, yet still prevents activity of AG in whorl 1, none of the three classes of homeotic genes would be active in whorl 1. The model would therefore predict the development of carpelloid leaves in these positions; this is what is observed in ap2-l flowers grown at 29 °C (Bowman et al. 1989).

First whorl positions in ap2-1 flowers grown at temperatures below 25 °C, however, are occupied by leaves with little or no carpel characteristics. In addition, in first and fourth whorl positions in ap2-1 ag-1 double mutants, and in all whorls in ap2-7-contaming triple mutant strains, leaves are found where carpelloid leaves are expected. The activity of AG is clearly reduced (or possibly absent) in the double and triple mutant flowers. Since the phenotype of the first whorl organs of ap2-1 flowers is nearly identical with that of the organs of the double and triple mutant flowers, the activity of AG must also be reduced in ap2-1 flowers. This implies a third function for the AP2 product, to repress an as yet-unknown homeotic activity similar to that of AG, which when expressed in leaves adds some carpel characteristics. Greater loss of AP2 function, as in ap2-2, allows this activity to function, thus producing carpelloid leaves, while a low level of AP2 activity, as in ap2-1, represses it (variably) and results in leaves with few or no carpel characteristics. Quadruple mutants for the unidentified gene and all three of the known homeotic paths would have true leaves in all floral organ positions, and this collection of gene classes would be sufficient for specifying all of the differences between vegetative and floral organs.

This proposal predicts either that the AP2 product has two or more separable domains of function, or that different levels of the AP2 product are required for its different functions. The staminoid petals in the second whorl of ap2-1 flowers are unlike those in the second and third whorls of ap2-2 ag-1 flowers (they consist of sectors of stamen tissue, sectors of petal tissue, and sectors that appear intermediate between petal and stamen tissue, Pruitt et al. 1987; Bowman et al. 1989). This implies variable repression of AG in the second whorl in ap2-1, and a variable ability of the AP2 product in this whorl to affect downstream genes. In the stamen tissue, the AP2 product appears not to accomplish either of its functions; in the petal tissue, it performs both of its functions; and m the hybrid sectors, it is repressing AG, but is not contributing to the differentiation process.

Unexplained complications

Mosaic organs and whorls

As stated previously, the proposed model does not address questions of how the homeotic genes affect organ number and organ position. Beyond this shortcoming, there are other observations relating to organ identity, which is within the realm of our model, that are not incorporated.

The presence of phenotypically mosaic organs in a number of the genotypes examined presents one aspect not readily explained by the model. Prominent examples are organs consisting of carpel and stamen sectors in the first whorl positions of ap2-2, ap2-8 and ap2-9 flowers. These ap2 alleles alter the number, size and shape of all four whorls of organ primordia, as well as the pattern of differentiation of the organs in the outer two whorls. The first whorl organ primordia from which the mosaic organs develop appear to be larger, and to involve a greater proportion of the flower primordium, than do the first whorl organ primordia in wild-type flowers (Fig. 2C,D). It could be that these organ primordia are large enough to encompass more than a single geographic whorl within the developing flower. The shape of these primordia is such that their margins could arise from cells that would normally be part of the second or third whorls while the central parts of the primordia arise from cells of the outer whorl. This would explain why the stamen sectors are always on the margins, and the carpel sectors always occupy the central portion of the phenotypically mosaic organs. A similar argument may be made for the phenotypically’ mosaic (mixed sepal and petal) inner whorl organs in ag-1, -2 and -3 flowers. This would suggest that the formation of fields specifying differentiation of cell types, and hence organ types, is to some degree independent of the formation of organ primordia.

The importance of organ primordium position within the developing flower is emphasized by the different fates of the medial and lateral first whorl organ primordia. As noted in this and in other studies (Komaki et al. 1988; Kunst et al. 1989) of ap2, the medial first whorl organs are generally carpelloid, while the lateral first whorl organs are quite leaf-like, even in the stronger alleles. In wild-type flowers, the lateral primordia are only slightly lower on the pedicel than the medial first whorl pnmordia (Smyth et al. 1990). By contrast, in ap2 flowers the lateral first whorl organ primordia are initiated much lower on the pedicel than the medial first whorl primordia. If the activity of the genes that specify floral organ identity is not present in the lower cells, which would normally give rise to the receptacle and pedicel, organ primordia arising ectopi-cally from these cells might be expected to differentiate, inappropriately, into leaf-like organs.

Secondary flowers

Another observation that does not immediately fit with the model is that of secondary flowers that develop in the axils of the lateral first whorl organs of singly, doubly and triply mutant flowers with the ap2-l genotype. The lateral first whorl organs in ap2-l resemble cauline leaves. They have stellate trichomes on their abaxial surfaces, leaf-like epidermal and internal cellular morphologies, and stipules at their base. These are all characteristics of leaves and not of floral organs. Another characteristic of Arabidopsis leaves, both radical (rosette) and cauline, is that each has an associated meristem in its axil. Thus, it may not be surprising that organs that have the identity of leaves develop meristems in their axils. For genuine radical and cauline leaves, the meristem is an inflorescence meristem. In ap2-l floral leaf-like organs, it is a floral meristem, indicating the possibility that meristem identity, like organ identity, can be determined independently of position.

Second whorl leaves

A final detail not easily explained by the model is the occurrence of leaf-like organs in the second whorl of ap2-l flowers grown at 16 °C (Bowman et al. 1989). Regardless of the activity of the product of the ap2-l allele, it is expected that the presence of wild-type PI and AP3 activities in this field B position would modify the leaf fate of these organs. It apparently does not, at least in some instances.

Interpretation of the ag fourth whorl and the problem of indeterminacy

The cells that would ordinarily give rise to the fourth whorl gynoecium in wild-type flowers form another flower in ag. The fourth whorl of organs in ag flowers may thus be interpreted as either a fourth whorl of sepals, replacing the fourth whorl carpels of wild-type flowers (the explanation used above), or as the first whorl of sepals in an internal flower. The number and positions of the organs support the second interpretation, while the identity is, in either case, consistent with our model. Therefore, the structure of ag flowers is most easily described as a reiterated pattern: (first whorl, second whorl, third whorl)n with no fourth whorl organs ever forming. Likewise, the structures of ag-1 pi-1 flowers and ap2-2 ag-1 pi-1 flowers could be described as the reiterated patterns of organs: (first whorl, second whorl)n and (medial first whorl)n, respectively. This interpretation does not affect the organ differentiation model presented, except to show that we may not know what the phenotype of AP2 expression in a fourth whorl organ might be.

The larger question of the nature of action of the wild-type AG product in suppressing indeterminate growth of the floral meristem is left unanswered. It seems that this activity could partially explain the absent organs in whorls 1 and 2 in ap2 mutants: the absence of AP2 activity in these whorls allows AG to act in them, which could then suppress cell division during their development. This is consistent with the observation that removal of the AG product from these ap2 whorls (in the ap2-2 ag-1 double mutants) partially restores organ number toward wild-type. The number of second and third whorl primordia in ap2-2 ag-1 flowers (average number of 7.6) is closer to the wildtype number (10) than the number found in flowers homozygous for ap2-2 alone (average 0.25).

Missing organs in mutant flowers result from the failure to initiate organ primordia

In several ap2 and pi alleles, organs fail to develop. For example, second and third whorl organs may be missing in ap2-2, ap2-8 and ap2-9 flowers, and second whorl organs are often absent in ap2-l/ap2-2 flowers and ap2-1 flowers grown at 29 °C (Bowman et al. 1989). In each of these instances, as well as in the pi alleles, there is no evidence of organ primordia at any stage of development in those positions lacking organs (Fig. 2C-F). Previous descriptions of the ap2-6 and ap2-7 alleles, however, (Kunst et al. 1989) suggested that second whorl organ primordia of flowers homozygous for these alleles form in wild-type positions and numbers, but fail to develop properly (ap2-7) or fuse to the first whorl organs (ap2-6). However, the outgrowths of cells described as second whorl organ primordia in ap2-6 and ap2-7 flowers may not be organ primordia, but rather stipules (due to the phylloid character of the outer whorl organs; see Fig. 11B of Kunst et al. 1989) or squamules, small filamentous outgrowths of cells that have been documented in other species of Brassicaceae (see Figs 5A,B and 11C,D of Kunst et al. 1989; Arber, 1931; Smyth et al. 1990) and apl-1 flowers (Irish and Sussex, 1990; D.R.S. and J.L.B. unpublished). Stipules arise early in development (stage 3–4 for first whorl organs) while squamules arise much later. Further analysis of developing ap2-6 and ap2-7 flowers should clarify this uncertainty.

Predicted spatial and temporal expression patterns

If the homeotic genes act autonomously, certain predictions can be made about their expression patterns. AP2 should be expressed in the outer two whorls and as early as stage two, prior to the appearance of the first whorl primordia (Bowman et al. 1989). Likewise, AG expression could be restricted to the inner two whorls. The expression of either AP3 or PI or both should be localized in the middle two whorls. AP3 should be expressed as early as stage five and should continue at least through stage seven and possibly much later (Bowman et al. 1989). The lack of third whorl primordia in pi-1 flowers suggests that its expression should start prior to stage five, the time at which the third whorl primordia arise. An important prediction is that the expression pattern of AG should expand to include all four whorls in an ap2 background and conversely the expression of AP2 should be in all whorls in an ag background. It should be noted that what is meant by expression is not restricted to the level of transcription since translational or post-translational control are plausible options. In addition, only one of the products needs be restricted to achieve the phenotypic results if one of the products is functionally inactive in the presence of the other

One of these genes, AG, has been recently cloned and encodes a putative transcription factor (Yanofsky et al. 1990). mRNA tissue in situ hybridization shows that its expression in wild-type plants is localized to whorls three and four (Yanofsky et al. 1990; Gary N. Drews, J.L.B., and E.M.M., submitted for publication). Furthermore, in an ap2-2 mutant background, the range of expression expands to include all whorls, supporting the hypothesis that AP2 negatively regulates AG in the outer whorls in the developing flower (G. N. Drews, J.L.B., and E.M.M., submitted for publication). Another flower homeotic gene, deficiens, which when mutated results in a phenotype similar to those of ap3 and pi, has been cloned from Antirrhinum (Sommer et al. 1990). This gene is predominantly expressed in petals and stamens although a low level of expression has been reported to occur in sepals and carpels (Schwarz-Sommer et al. 1990).

The timing of initial AG and AP2 expression could play a role in their localization. Tissue in situs to mRNA show that AG expression is not detectable until stage three, when the first whorl organ pnmordia become morphologically distinct from the remainder of the developing flower. In contrast, the tsp of the ap2-l allele encompasses stages two through four, prior to and during the formation of the first whorl primordia, suggesting that the initial expression of AP2 precedes that of AG. Since the formation of the four whorls of floral organ pnmordia is a sequential process, it is likely that the expression patterns of the genes responsible for pattern formation in the developing flower are influenced by this sequential nature. For example, AP2 could initially be expressed in cells that will give rise to the outer two whorls of organs prior to and during the time of their formation. This would preclude the expression of AG in these whorls. AP2 expression would then be curtailed in the remaining floral meristem due to either loss of positive regulation by some factor that was responsible for its expression in the outer two whorls or negative regulation by a newly expressed factor, such as AG. In this case, AG would be expressed in those cells that will give rise to the third and fourth whorl organs precluding AP2 expression in the inner two whorls. The spatial regulation of AP3 and PI could be attained by having their expression commence only after the first whorl is formed and having a gene such as SUPERMAN (Bowman and Meyerowitz, 1991; Meyerowitz et al. 1991) negatively regulate their expression in the fourth whorl. Thus, mutations in AP2 would be expected to have the most dramatic effects since it is one of the first genes expressed in sequential but overlapping gene pathways specifying both cellular identities and organ primordium patterns.

A candidate for a gene responsible for the regulation of the homeotic genes is leafy (Haughn and Somtner-ville, 1988; Detlef Weigel, John Alvarez, and D.R.S., unpublished). When this locus is mutated, the floral meristem behaves as if it were an inflorescence meristem. Each floral meristem produces a number of cauline leaf-like organs in a phyllotactic spiral; the later ones produced are often carpelloid. This phenotype is similar to that of the triple mutant ap2-2 pi-1 ag-1 flowers in which all organs are carpelloid leaves but, in the case of the triple mutant strain, the organs are arranged in a radial phyllotaxy. Other genes must be involved as well, to produce the complex patterns of expression expected of the later-acting homeotic genes, and the change in phyllotaxy from spiral to whorled.

Evolutionary considerations

Finally it should be noted that mutations similar to those described here exist in many other species of plants. For example, the agamous phenotype, which is commonly referred to as a double flower, has been described in Matthiola (Dodoens, 1568: see Saunders, 1921), Cheiranthus (Masters, 1869), Petunia (Sink, 1973) and Antirrhinum (Carpenter and Coen, 1990; Schwarz-Sommer et al. 1990) as well as many others (see Penzig 1890-4; Meyer, 1966; Reynolds and Tampion, 1983; Meyerowitz et al. 1989; for reviews). ap2 homologues have been reported in Capsella (Dahlgren, 1919; Shull, 1929) and Antirrhinum (Carpenter and Coen, 1990; Schwarz-Sommer et al. 1990) and ap3fpi homologues have been described in Cheiranthus (Sirks, 1924; Nelson, 1929) and Primula (Brieger, 1935) in addition to Antirrhinum (Stubbe, 1966; Sommer et al. 1990; Carpenter and Coen, 1990). That numerous mutants homologous to those described here occur in other plant families suggests that the mechanisms governing flower development in Arabidopsis are likely to operate in many flowering plants. When genetic experiments similar to those reported here have been done with other species, direct comparisons of the actions and interactions of homeotic genes in these species will be possible, and an assessment can be made of the degree to which these mutations are homologous with those of Arabidopsis.

We thank our colleagues, Laura Brockman, Caren Chang, Gary Drews, Bruce Hamilton, Tom Jack, Leslie Sieburth, Alex van der Bhek and Detlef Weigel for insightful discussions and critical review; Ken Feldmann for providing the ag-2 allele; and Pat Coen of the Electron Microscope Facility at Caltech for technical advice. This study was assisted by National Science Foundation grant DCB-8703439 to E.M.M. J.L.B. is partially supported by training grant 5T32-GM07616 of the National Institutes of Health.

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