The Plant Cell, Vol.

1,37-52, January 1989, © 1989 American Society of Plant Physiologists

Genes Directing Flower Development in Arabidopsis

John L. B o w m a n , David R. Smyth, 1 and Elliot M. M e y e r o w i t z 2
Division of Biology, 156-29, California Institute of Technology, Pasadena, California 91125

We describe the effects of four recessive homeotic mutations that specifically disrupt the development of flowers
in Arabidopsis thaliana. Each of the recessive mutations affects the outcome of organ development, but not the

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location of organ primordia. Homeotic transformations observed are as follows. In agamous-1, stamens to petals; in
apetala2-1, sepals to leaves and petals to staminoid petals; in apefala3-1, petals to sepals and stamens to carpels;
in pistillata-1, petals to sepals. In addition, two of these mutations (ap2-1 and pi-1) result in loss of organs, and
ag-1 causes the cells that would ordinarily form the gynoecium to differentiate as a flower. Two of the mutations
are temperature-sensitive. Temperature shift experiments indicate that the wild-type AP2 gene product acts at the
time of primordium initiation; the AP3 product is active later. It seems that the wild-type alleles of these four genes
allow cells to determine their place in the developing flower and thus to differentiate appropriately. We propose
that these genes may be involved in setting up or responding to concentric, overlapping fields within the flower
primordium.

INTRODUCTION

Flowers develop from groups of undifferentiated cells that These particular mutations were chosen for detailed
grow from the flanks of shoot apical meristems. The cells study because each appears to cause cells in the devel-
in these floral primordia divide and then differentiate into oping flower to misinterpret their position, and thus differ-
appropriate numbers of floral organs, in appropriate entiate into inappropriate cell types. At the same time,
places. During this process of development, each cell must none of the mutations appears to cause any abnormal
somehow determine its position relative to others, and phenotype outside of the flower. The adult phenotypes of
must differentiate accordingly. Nothing is known about the some alleles of all of them have been described before,
mechanisms by which the cells of a developing flower although not in detail (Koornneef et al., 1983; Pruitt et al.,
establish their positions and subsequently give rise to 1987; Bowman et al., 1988; Haughn and Somerville, 1988).
appropriate cell types. Several processes do not seem to In addition to a detailed description of the adult phenotype,
be involved: there is no cell migration in higher plants, and we give here a description of the development of each
the totipotency of many higher plant cells indicates that mutant type, based on scanning electron microscopy.
floral primordia do not rely on deposition of positional cues Furthermore, we show that mutant alleles of two of the
in the egg cell, as is sometimes the case in aspects of genes are temperature sensitive, and we determine the
animal development. As an approach to revealing the temperature-sensitive developmental stage. Finally, the
mechanisms by which cells in developing flowers choose phenotypes and development of double mutants are de-
an appropriate developmental fate, we are studying genes scribed as a means of understanding the interactions of
whose wild-type products seem to play a central role in the gene products.
these mechanisms (Pruitt et al., 1987; Bowman et al., As a background to the descriptions of these mutant
1988). plants, we must describe briefly the appearance of wild-
In this paper we describe four homeotic mutations in the type flowers. A mature Arabidopsis flower is typical of the
flowering plant Arabidopsis thaliana, each of which ap- flowers of plants in the Brassicaceae. It is composed of
pears to affect fundamental processes in floral develop- four concentric whorls. The first whorl is occupied in wild-
ment. The mutations are agamous (ag), apetala2 (ap2), type flowers by four sepals. We will ignore the two-whorl
apetala3 (ap3), and pistillata (pi) (Koornneef et al., 1983). interpretation of the four sepals in the Brassicaceae (see
All of them are recessive mutations in single genes. Lawrence, 1951) because there seems no compelling rea-
son to treat the two pairs of sepals differently, and because
1Permanent address: Department of Genetics, Monash we wish to avoid the old controversies about whether the
University,Clayton, Victoria 3168, Australia. lateral or the medial sepals belong to the outermost whorl
2To whom correspondence should be addressed. (Arber, 1931). Inside and alternate to the sepals are four
38 The Plant Cell

petals, which occupy the second whorl. Historically, the first appear on the rim of the cylinder at the beginning of
stamens of flowers in the mustard family have been con- stage 11. This is also the stage at which the lateral
sidered as two whorls, the outer containing the two lateral nectaries appear at the base of the lateral filaments; the
stamens, and the inner the four medial ones (see Law- development of nectaries at the base of the other filaments
rence, 1951). Because no studies have revealed funda- occurs later. Stage 12 begins when petals reach the height
mental differences between different stamens, we will for of the long stamens. Figure 1A shows a mature Arabidop-
convenience and simplicity refer to the region containing sis flower; Figure 2A shows some of its early develop-
the stamens as the third whorl. The center of the flower is mental stages. A detailed description of early flower de-
occupied by a gynoecium made up of an ovary that con- velopment in Arabidopsis is being prepared (D.R. Smyth,
tains two chambers separated by a septum. The ovary is J.L. Bowman, and E.M. Meyerowitz, manuscript in
topped by a short style and a papillate stigma. Within the preparation).
ovary there develop approximately 50 ovules, attached in

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rows to the margins of the carpels. The region occupied
agamous (ag)
by the gynoecium in the wild-type flower will be referred
to as the fourth whorl. In describing the mutants, we will
agamous flowers consist of many sepals and petals, and
use whorl to indicate a region of the flower, regardless of
of chimeric organs consisting partly of sepal and partly of
the nature of the organs contained within it.
petal tissue. There are no stamens or carpels. The mutant
The flowers develop in a raceme, so that a single stem
flowers have an outer whorl of four sepals, then a series
can have a series of flowers in different stages of devel-
of 10 petals, and in the place of the gynoecium a variable
opment, from primordia at the top to mature fruits nearer
number of sepals, petals, and intermediate organs (Figure
the bottom. The development of individual flowers is much
1 B). The mutant allele of AG used (Koornneef et al., 1980)
like that described for Cheiranthus cheiri by Payer (1857),
is here designated ag-1; its locus is on the fourth chro-
and Brassica napus by Polowick and Sawhney (1986). The
mosome (Koornneef et al., 1983). Observations of devel-
earliest and latest stages of flower development in Arabi-
oping ag flowers make clear the developmental basis of
dopsis have been described (Vaughan, 1955; MUller,
the phenotype: The sepals form normally in the first whorl;
1961).
the primordia of the second and third whorls also form in
their wild-type positions. The remaining cells, which would
normally develop into the ovary, behave, however, as if
RESULTS they constituted a new floral primordium (Figure 2B). Four
new sepals form at its margins, and apparent petal and
stamen primordia develop inside of them. The central cells
Development of Wild-Type Flowers of this second flower also develop as a new flower, which
itself has a new flower develop inside of it, and so on for
Scanning electron microscope observations of developing enough rounds to result in a mature flower with more than
A. thaliana flowers has allowed their early development to 70 organs.
be divided into 12 stages. Flower initiation begins when In addition to the development into new flowers of the
the cells that will develop into the flower form a buttress cells that would ordinarily form an ovary, the third whorl
on the flank of the florally induced shoot apical meristem primordia of each flower develop into petals, not stamens;
(stage 1). As this group of cells grows, an indentation their development is similar in its time course with that of
arises that separates it from the adjacent meristem, at petals, and the organs they form are petals. Although the
which time stage 2 begins. After this, sepal buttresses number of primordia that form petals in the outer flower is
form on the primordium (stage 3 begins) and grow to form a uniform 10, the inner flowers have irregular numbers and
distinct ridges (stage 4). The abaxial and adaxial (medial) positions of primordia that will become petals, and thus
sepals form before the lateral ones. The primordia of petals irregular numbers and positions of the inner petals. One
and stamens then appear, and the continued growth of other irregularity is also apparent. The organs that develop
the medial sepals causes them to meet and cover these at the margin of each of the internal flower primordia are
developing inner organs, marking the start of stage 5. not perfect sepals, instead they are mosaics of sepal and
Stage 6 begins when the lateral sepals meet. The primordia petal tissue. The mosaic sectors always extend from the
of the petals do little until later in flower development; the base to the apex of the organs, with sepal tissue in the
stamens develop first. Stage 7 begins when the filament center and petal tissue at the margins (Figure 3). Table 1
and anther precursors become distinct; stage 8 when summarizes the ag phenotype.
Iocules appear in the anthers. Petal elongation accelerates
as stage 9 starts; the length of the petals equals that of
the short stamens when stage 10 begins. During these apetala2 (ap2)
stages, the gynoecium is developing from the cells interior
to the stamens: An initial dome of cells becomes a cylinder apeta/a2 is a fourth chromosome gene, mapping more
as the cells of the periphery grow; the stigmatic papillae than 25 centimorgans from ag (Koornneef et al., 1983).
 Flower Development in Arabidopsis 39

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Figure 1. Phenotypes of Wild-Type and Mutant A. thaliana Flowers.

(A) Wild-type.
(B) agamous.
(C) apefa/a2.
(D) apetalaS.
(E) pistillata.
The plants were grown at 25°C. Bar = 1 mm.

The original ap2 mutant allele (Koornneef et al., 1980), two whorls. Plants grown at 25°C have an outer whorl
which we designate ap2-1, is temperature sensitive, with consisting of four organs resembling cauline leaves, rather
different phenotypes at different temperatures (Tables 1 than four sepals (Figure 1 C). That they are leaflike is shown
and 2). At all temperatures, the effects are on the outer by the presence of stellate trichomes, which are charac-
40 The Plant Cell

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Figure 2. SEM Micrographs of Early Flower Development in Wild-Type and Mutant Arabidopsis Plants Grown at 25°C.
The first (lefthand) panel in each series displays the apical meristem and stages 1 through 4 of flower development, with the exception of
(B), which shows through stage 6. The second panel shows stage 6, the third panel stage 8, and the fourth panel displays flowers near
maturity. In the second and third panels, one to three outer whorl organs have been removed to reveal the inner whorls. In the fourth
panel, outer and in some cases (B,D,E) second whorl organs haye been removed.
(A) Wild-type. Sepal (se), petal (p), medial stamen (mst), and lateral stamen (1st) primordia, and the gynoecial cylinder (g) are indicated.
(B) agamous. Nested flowers are visible in the third and fourth panels.
(C) apetala2. A stipule (sp) is indicated in the third panel. A trichome (t) at an early stage of development is also noted. The appearance
of stigmata on the outer whorl organs precedes their appearance on the gynoecium, as seen in the fourth panel.

teristic of leaves (sepals have few, simple trichomes), not in genuine sepals (Figure 2C). In two respects these
appearing as early as stage 7 on both sides of the organs. organs are not leaflike: they have the long (>100 ^m)
These trichomes are more dense on the abaxial surface; epidermal cells that are characteristic of sepals, but not
in genuine cauline leaves they are more dense on the leaves, on their abaxial surface. In addition, they often
adaxial. In addition, these organs senesce in a way un- have stigmatic papillae at their tips, revealing a slight
characteristic of sepals, in that they do not yellow and fall gynoecial transformation. The frequency with which stigma
off shortly after anthesis. Furthermore, the early develop- tissue is seen at the tips of these organs depends on the
ment of the organs of the ap2 outer whorl is characterized position of the flower in the inflorescence, with later flowers
by the presence of stipules, present in cauline leaves but showing papillae more often. In addition, stigmatic papillae
 Flower Development in Arabidopsis 41

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Figure 2 (continued).
(D) apetalaS. Note the delayed development of the second whorl primordia in the third panel, even though they differentiate into sepals
rather than petals (fourth panel).
(E) pistillata. The third whorl primordia fail to appear. Bar = 10 ^m in the first three panels of each series; bar = 100 ^m in the fourth
panel.

occur more frequently on the medial than on the lateral

organs. These papillae are first seen in stage 10 of devel-
oping ap2 flowers, prior to the appearance of the stigma
on the gynoecium.
The second whorl of ap2 flowers grown at 25°C shows
transformation of petals toward stamens. The transfor-
mation is seldom complete, with most organs having fea-
tures of both petals and stamens; the degree of transfor-
mation increases with increasing age of the inflorescence
(Figure 4). The intermediate and the most staminoid organs
contain pollen grains in locules; only the most staminoid
dehisce.
At 16°C, the outer whorl of ap2-1 homozygous flowers
is the same as at 25°C; conversion of sepals to leaves
with stigmatic papillae at their tips. At the lower tempera-
ture, however, there is very little stigmatic tissue evident.
The second whorl is quite different than at the higher
temperature, exhibiting in the first flowers on a stem an
Figure 3. Distinct Petaloid and Sepaloid Regions Are Visible in a outward rather than an inward homeosis: the organs range
Mosaic Organ of an agamous Flower. from petals to leaflike structures (Figure 4, Table 2). Or-
The transition between the two types of tissue is usually abrupt gans intermediate between petals and leaves may contain
with a zone of one to three cells of intermediate phenotype. a distinct, longitudinal boundary between green and white
Bar = 30 ^m. regions, but the white regions have epidermal cells that
42 The Plant Cell

Table 1. Summa~ofPhenotypes
Fi~tWhod SecondW h o r l ThirdWhod Fou~hWhod
Landsbergerecta
Wild-Type(wt) Sepals Petals Stamens Carpels
agamous 16-25°C wt wt Petals Flower development
repeats
apetala2 16°C Leaves wt or slightly wt wt
phylloid petals
25°C Stigmoid Staminoid petals wt wt
leaves
29°C Carpelloid Absent wt wt
leaves

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apetala3 16°C wt Sepaloid petals wt wt
25°C wt Sepals Carpelloid wt
stamens
29°C wt Sepals Carpels wt
pistillata 16-25°C wt Sepals Absent Extra carpels

show characteristics of both petal and leaf cells (Figure 5). First, simple temperature shifts were done. Plants were
Even the organs most resembling petals are seen to have taken from an incubator at 16°C and transferred to one
stomata, which are not ordinarily found on petals. This maintained at 29°C, or vice versa. Since each plant had
outward transformation decreases in later flowers, with many flowers at many different stages of development,
those flowers after the first 10 showing a slight inward the effect of a shift on the organs of the second whorl was
transformation, as at higher temperatures. recorded at all stages of floral development. As seen in
At 29°C, the outer whorl consists of leaves with a the data in Figure 6, A and B, temperature shifts in either
greater transformation toward gynoecial tissue than at direction indicate that the function of the AP2 product is
lower temperatures. Stigmatic tissue occurs at the tip of no later than the developmental period from stage 2 to
almost every organ and may extend down the lateral stage 4. Later than this, temperature shifts have no effect.
margins. Naked ovules develop on one (13 out of 136 This developmental time extends from just prior to the
organs counted) or both (2 out of 136 organs) margins; appearance of the outer whorl primordia to the time just
this occurs primarily on the medial organs. The most before the appearance of the second whorl pdmordia.
carpelloid organs resemble solitary unfused carpels, but The second type of temperature shift experiment done
with the stellate trichomes characteristic of leaves on their was a temperature pulse, in which plants at 16°C were
abaxial surface. The second whorl organs of flowers grown shifted to 29°C for 48 hr, and then shifted back to the
at 29°C either fail to develop at all or are transformed lower temperature, or, conversely, plants at 29°C were
more toward stamens than at 25°C. As at 25°C, the extent
of staminody of these organs increases with increasing
inflorescence age. The effects of temperature on the de-
velopment of the organs of the second whod are detailed Table 2. Phenotypes of SecondWhorl Organsinap2 Flowersa
in Table 2.
16oc 25oc 29°C
Observations of developing flowers indicate that the
failure of an organ to appear in the second whorl is a result % % %
of a failure of the organ primordium to initiate development. Absent <1 29 73
At no temperature does there appear to be a correlation Stamen 0 0 3
between the phenotype of a second whorl organ and its Deformed 0 2 5
position within the whorl. Nearly wild-type and almost Stamen
Filament 0 0 3
completely transformed organs may develop in adjacent <1 24 7
Petaloid Stamen
positions. The organs of the second whorl, regardless of Staminoid Petal 6 37 10
their eventual developmental fate, develop on a time Petal 82 9 0
course characteristic of wild-type petals: they develop after Phylloid Petal 6 0 0
the organs of the other whorls of the flower. Petaloid Leaf 3 0 0
The fact that the ap2-1 allele is temperature sensitive Cauline Leaflike 2 0 0
allows temperature shift experiments to reveal the time at The second whorl organs of the first 10 to 14 flowers produced
which the wild-type AP2 gene product is active in flower on at least four plants were scored and classified according to
development. Two types of experiment were performed. the phenotypes described in Figure 4.
 Flower Development in Arabidopsis 43

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Figure 4. SEM Micrographs of Organs Observed in the Second Whorl of apefa/a2 Flowers.
Petals and leaflike organs are common at 16°C, stamen-like petals typical at 25°C, and petal-like stamens or no organ occurring at 29°C
(Table 1). To allow comparison, the intermediate forms were categorized.
(A) Organs with no trace of white tissue were classified as cauline leaflike, whereas those with a small amount (B) were termed petaloid
leaves.
(C) Mostly white organs or those all-white organs possessing trichomes were classified as phylloid petals.
(D) Morphologically wild-type petals were typical at 16°C.
(E) White, petal-shaped organs possessing rudimentary locules were termed staminoid petals.
(F) Those organs classified as petaloid stamens were shaped like stamens but with some white petal tissue, usually near the top of the
organ. Misshapen stamens and filaments lacking anthers were observed at a low frequency.
(G) Morphologically wild-type stamens occur at a low frequency at 29°C.
Outer surfaces are shown in (A), (B), and (C); inner surfaces in (D), (E), (F), and (G). Bar =100 ^m.

shifted to 16°C for 90 hr (a developmental time at 16°C range from apparently normal stamens to carpelloid sta-
equivalent to 48 hr at 29°C), and then returned to 29°C. mens to normal-appearing, but unfused, carpels, as shown
Flowers developed to show the phenotype corresponding in Figure 7. The degree of carpellody increases with in-
to the temperature they experienced at stage 2 to 4, creasing temperature and increasing age of the inflores-
indicating that the AP2 product acts no earlier and no later cence, and the organs replacing the two lateral stamens
than this period of development. That ap2 flowers held at are less carpelloid than those replacing the four medial
16°C for a brief period in their development can form stamens (Table 3). These organs, when fully carpelloid,
petals shows that the AP2 product need only be active for have up to five well-developed ovules along each margin
this brief period to specify the initiation and differentiation and are capped with stigmatic papillae. The appearance
of the organs of the second whorl. The temperature- of their epidermal cells in the scanning electron microscope
sensitive period, from stages 2 to 4, lasts approximately is identical with the appearance of the epidermal cells of
75 to 90 hr at 16°C, and only 30 to 50 hr at 29°C. genuine ovaries. In some flowers two or three of the six
carpelloid organs may fuse to form a hemispherical struc-
ture. Intermediate organs are mosaics, consisting of a
apefa/a3 (ap3) patchwork of sectors with epidermal features of either
stamens or carpels. A single organ may possess both
The ap3-1 allele of the AP3 gene (Bowman et al., 1988), ovules and pollen.
like ap2-7, is a recessive temperature-sensitive mutation. The development of ap3 flowers at 25°C is morpholog-
This third chromosome mutation, like ap2-7, also causes ically identical to wild-type until stages 7 to 8, when the
transformations in two adjacent whorls, but they are the filament and anther would normally become distinct. At
second and third, rather than the first and the second, this time it becomes clear that the organs of the third whorl
ones (Table 1). At 25°C or 29°C, ap3-7 homozygotes are not producing these structures, but are elongating
develop flowers in which the organs of the second whorl vertically and forming the epidermal cell files seen on
are sepals (Figure 1D), indistinguishable from wild-type normal gynoecia (Figure 2D). The stigmas of the isolated
sepals except by their slightly smaller size. Despite their carpels appear slightly earlier than those of the the central
transformation, these organs develop in the positions, and gynoecium, with ovule formation beginning before this
on a time course, characteristic of petals. The organs of stage.
the third whorl of ap3 flowers grown at or above 25°C At 16°C, plants homozygous for ap3-7 have a second
44 The Plant Cell

(A) apetala2 16 C to 29 C

absent 65 13 46 1
stamen 1
deformed stamen 1 1 6 2
petaloid stamen 1 6 3
staminoid petal 1 1 10 2
petal 1 22 30 39
phylloid petal 1 1 18
petaloid leaf 4 3 4

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leaf-like 1 2 4 1

stage of flower m b p se se + bud

at time of shift (1) (2) (3,4) (4,5)
(6+)

(B) apetala2 29 C to 16 C

absent I 9 11 9 8
stamen
deformed stamen 1 6
petaloid stamen 1 2 5
staminoid petal 2 1 4 3 4 4

Figure 5. Higher Magnification (bar = 30 ^m) of the Petaloid Leaf petal 43 14 15 1 1 5

Shown in Figure 4B Showing the Patches of Green Leaflike Tissue phylloid petal 1
with Wild-Type Leaf Epidermal Morphology and the White Tissue petaloid leaf 1 1 2
with an Epidermal Morphology Intermediate between that of Pet- leaf-like 1
als and Leaves.
stage of flower m b P se se + bud
A similar situation is observed in staminoid petals: patches of at time of shift (1) (2) (3,4) (4,5) (6+)
stamen tissue with nearly wild-type stamen epidermal morphology
are adjacent to white petal-like tissue with an epidermal morphol-
ogy intermediate between that of petals and stamens.
Figure 6. Temperature-Sensitive Period (TSP) of the Phenotype
of the Second Whorl Organs of apetala2-l Flowers.

whorl that is nearly wild-type, with organs that are partly Since each inflorescence consists of flowers at many stages of
or completely white and with the smooth margins of petals, development, shifting a small number of plants provides data on
all stages of flower development. Plants were either germinated
rather than the uneven edges of a sepal. The organs are
and grown at the permissive (16°C) temperature and shifted to
not completely normal petals: they fail to reach normal the restrictive (29°C), or the converse shift was performed. The
petal size, and their epidermis is like that of sepals, con- developmental stage of all flowers at the time of the shift was
sisting of stomata and irregularly shaped cells without the noted. Because all stages defined using SEM are not distinguish-
radial cuticular thickenings of petal epidermal cells. The able in the dissecting microscope, the following stages were used:
organs in the third whorl of ap3 plants grown at 16°C all those flowers that were not initiated at the time of the shift were
develop as stamens; at this temperature the flowers can placed in the meristem (m) stage. Stages 1 and 2 were defined
self-fertilize and produce homozygous seed. as buttress (b) and primordia (p), respectively. Stages 3, 4, and 5
Figure 8, A and B, summarizes temperature shift exper- were combined into two stages: sepals (se) and sepals-plus (se+).
The sepals stage has only sepals initiated, whereas the sepals-
iments with ap3-1 homozygotes, performed to determine
plus stage has third whorl primordia initiated but the sepals have
the temperature-sensitive period of the third whorl in these
not yet enclosed the bud. The remaining flowers (stage 6 on) were
flowers. This period is later than with ap2-7. A shift up as simply classified as buds. When the flowers had matured, the
late as stage 5 can cause complete conversion of third second whorl organs of each were scored as outlined in Figure 4.
whorl organs to carpels, and there is a partial conversion The numbers are the number of organs of each type.
after a temperature shift up as late as stage 7 and perhaps (A) Fourap2-7 plants shifted from 16°C to 29°C.
even 8. A shift down, from 29°C to 16°C, has a clear (B) Four ap2-7 plants shitted from 29°C to 16°C. The TSP of the
effect until after stage 6. Thus, the AP3 gene product second whorl appears to encompass stages 2, 3, and part of 4.
 Flower Development in Arabidopsis 45

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Figure 7. SEM Micrographs of the Inner Surface of Organs Observed in the Third Whorl of apetalaS Flowers.
(A) Morphologically wild-type stamens are observed at 16°C; carpel-like organs (E) are typical at 29°C. Mosaic organs possessing
characteristics of both occur at intermediate temperatures (Table 3). The mosaic organs were categorized to permit comparisons of
phenotypes under different growth conditions. If stamens were capped with stigma but no other deformities of the anther were present,
or if the anther was misshapen but not carpelloid, the organs were classified as deformed stamens (not shown).
(B) Those with the shape of stamens, with prominent locules, but capped with stigma and showing developing ovules at the base of the
outer locules were termed carpelloid stamens. The most developmentally advanced ovules occurred at the base of the anthers, whereas
less well developed ovules occurred farther up.
(C) Organs shaped like carpels, capped with stigmatic papillae, and possessing ovules, but with remnants of locules and a filamentous
base were termed staminoid carpels.
(D) Filaments with no anther but sometimes topped with stigma may form at higher temperatures.
Bar = 100 ^m in (A), (B), (C), and (E), and 30 ^m in (D).

appears to be effective in specifying the fate of third whorl more advanced developmental stages at the time of the
organ primordia up to the time when they begin their shift than those in which third whorl effects are seen. Thus
differentiation. The results of temperature pulse experi- again, the AP3 product acts in flowers up to the time when
ments are in accord with those of the shift experiments. If differentiation of affected organs begins.
plants are grown at 16°C, shifted to 29°C for 54 hr, and
then returned to 16°C, flowers that were in stages 5 up
to and past bud closure (stage 6 and perhaps beyond) are pistillata (pi)
affected. The converse experiment, in which plants are
changed from 29°C to 16°C for 123 hr (a developmental PI is a gene on chromosome 5; the recessive mutant allele
time at 16°C roughly equivalent to 54 hr at 29°C), and pi-1 (Koornneef et al., 1983) affects the development of all
then returned to 29°C, flowers in stages 5 and 6 are floral organs except the sepals. The organs of the second
affected. TheX>P3 gene product thus acts much later than whorl develop as small sepals rather than petals, the
that of AP2. The AP3 temperature-sensitive period lasts organs of the third whorl do not develop at all, and the
approximately 40 to 50 hr at 29°C, and 80 to 100 hr at central gynoecium develops abnormally (Table 1). The
16°C. mature flowers thus consist of two outer, alternate whorls
The temperature-sensitive period of the second whorl in of sepals surrounding a large club-shaped gynoecium,
ap3 flowers is more difficult to specify since the organs of usually composed of more than two carpels (Figure 1 E).
this whorl are not completely converted to wild-type at The ovary may exhibit unfused carpel margins and vertical
16°C. Nonetheless, in a shift-down experiment, the 16°C filamentous appendages fused to its outer surface.
phenotype of the organs of the second whorl was ob- The development of pi flowers is indistinguishable from
served in flowers that were at the same or even slightly that of wild-type until the time of the appearance of the
46 The Plant Cell

Table 3. Phenotypes of Third Whorl Organs in ap3 Flowersa Two general classes of interaction were observed: com-
binations in which the effects of the two mutations ap-
25°C, peared purely additive (ag ap3; ag pi) or close to additive
Medial Only 29°C'
16°C, All (ag ap2), and combinations in which phenotypes were
Positions 1-7 b 8-15 b Lateral Medial observed that are not seen in plants homozygous for only
% one of the mutations (ap2 ap3; ap2 pi). ap3 pi mutant
Carpel 0 0 29 6 73 plants have not been studied: the crosses to produce them
Filament 0 0 0 8 8 gave no plants that looked different from single homozy-
Staminoid Carpel 0 0 40 9 4 gotes, indicating the possibility that the double mutant
Carpelloid Stamen 0 9 28 25 7 phenotype is identical to that of one of the single mutants.
Deformed Stamen 0 37 3 38 5 The additive combinations all included agamous, ag ap3
Stamen 100 53 0 0 0 flowers grown at 25°C have the multiplication of organs

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Absent 0 1 1 15 4
Third whorl organs of the first 15 flowers produced on at least
four plants were scored and classified according to the outline in
Figure 7. (A) apetala3 16°C to 29°C
b Numbers refer to position of flowers within the inflorescence,
with 1 being the first flower produced.
absent 9 2 2
carpel 30 5 13
filament 5 8
staminoid carpel 1 I
primordia of the second and third whorls (stage 5). In pi carpelloid stamen i0
mutants the second whorl primordia appear in the appro- deformed stamen 6

priate place at the correct time, but the third whorl primor- stamen 36

dia are not seen (Figure 2E). The gynoecium forms from
the cells encircled by the second whorl primordia, so that stage of m/b/p se se+ ist 2nd, 3rd older
it appears that the cells that would ordinarily be in the third flower at bud buds buds
time of shift
whorl are instead incorporated into the developing ovary. (1,2) (3,4) (4,5) (6) (7,8?) (8+)
Gynoecium development proceeds with characteristically
rapid vertical growth of the periphery of the central dome
of the flower primordium, but the diameter of the cylinder
(B) apetala3 29°C to 16°C
that is formed is much greater than in wild-type. Growth
of the cylinder can soon be seen to be irregular, with extra
carpels often forming and regions sometimes lagging be- absent 2 2 1
hind in vertical growth. One to four filamentous appen- carpel 1 2 16
dages emerged from the surface of the gynoecium in 56% filament 1
of 75 flowers examined; these appear to arise at the margin staminoid carpel 2 15
between carpels and can be fused to the ovary at their carpelloid stamen 2 4 18
base or along their entire length. These develop late, from deformed stamen 3 1
the wall of a developmentally advanced gynoecium. The stamen 74 36 21 18 4

mature gynoecium has two to five apparent carpels, with

an average of 2.7 (206 carpels counted in 75 flowers). The stage of flower m/b p se se+ ist bud
style and stigma are expanded in correlation with the at time of shift bud
{i) (2) {3,4) (4,5) (6) (6~)
increase in carpel number.
While the abnormal ovary is forming, the pdmordia of Figure 8. The TSP of the Third Whorl of apetala3Flowers.
the second whorl differentiate just as in flowers of ap3-1
homozygotes at restrictive temperatures: they differentiate The procedures outlined in Figure 6 were followed with the
exception that the youngest enclosed bud (1st bud), as well as
into sepals, but following a developmental time course
the next two youngest buds in the 16°C to 29°C shift (2nd, 3rd
characteristic of petals (Figure 2E). buds), were tallied separately from the rest of the older enclosed
buds. The stages of these buds were inferred from SEM of
dissected buds of other inflorescences. Medial third whorl organs
Double Mutants
were classified as described in Figure 7.
(A) Two ap3-1 plants shifted from 16°C to 29°C.
Plant lines homozygous for pairs of the four mutations (B) Four ap3-1 plants shifted from 29°C to 16°C. The TSP of the
described were constructed to examine the epistatic rela- third whorl in ap3 flowers extends from stage 5 up to and possibly
tions of these genes and their phenotypic interactions. including stage 8.
 Flower Developmentin Arabidopsis 47

and indeterminate growth of ag flowers, but instead of of 109 counted) or be fused to the gynoecium (2 out of
whorls of sepals and petals, as in ag homozygotes, they 109).
have whorls of sepals only (Figure 9A). ag pi flowers (Figure The phenotype of ap2 ap3 double mutants (Figure 9E)
9B) also consist of many whorls of sepals. The outer two is also nonadditive and dependent on temperature. At
whorls are initiated correctly, but the third whorl primordia 25°C, the four organs of the outer whorl are similar to
fail to appear, as in pi flowers. The remaining tissue follows those in ap2 flowers, but with a greater degree of carpel-
the pattern of indeterminate growth characteristic of ag, Iody, in that nearly all have stigmatic papillae, and many of
with nested internal flowers. those in medial positions have ovules on their margins.
ag ap2 double mutants grown at 25°C (Figure 9C) have Early flowers have second whorl organs resembling leaves,
an overall morphology characterized by indeterminate but most also have rudimentary Iocules, and are thus
growth and mosaic organs, as do ag single mutants. The staminoid. This is unlike the second whorl organs in the
identity of the organs is altered from that in ag, however. ap2 pi double mutants, which show no staminody. In later

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All organs and sectors of organs that would be sepaloid in flowers the second whorl organs show increased carpel-
ag flowers are leaflike (with no stigmatic tissue) in ag ap2 Iody, so that they can consist of a mosaic mixture of leaf,
double mutant flowers. The leaflike structures are dense stamen, and carpel. After the tenth flower on a stem,
with trichomes but lack any sign of the stigmatic papillae subsequent flowers usually lack all second whorl organs.
found on the leaflike organs of ap2 flowers. The remaining The positions usually occupied by the four long medial
organs and sectors, which would be petals in an ag single stamens are either filled by solitary carpels (51% in 15
mutant, are short, fleshy structures similar in shape to scored flowers), by anthers (9%), or by filaments without
rudimentary petals, but occasionally possessing the exter- anthers (10%); the remaining 30% of the positions had no
nal ridges that cover the Iocules of wild-type anthers, and organs. Twenty-eight percent of the positions usually oc-
showing the yellow-green color of developing anthers. At cupied by the two short lateral stamens had carpels; 19%,
16°C the ag ap2 double mutants still have leaves in place mixed stamen/carpels; and 53%, no organ. Examination
of sepals, but the remaining organs are petals. One feature of developing flowers shows that missing organs result
of the double mutant not usually seen in ag alone is a from failure of formation of an organ primordium.
greater degree of pedicel elongation between the nested At 16°C, the first whorl of ap2 ap3 double mutants is
flowers. made of four leaves, and the second whorl is made of
Turning to the nonadditive interactions, double mutant green organs that appear to be leaves, but with far fewer
flowers homozygous for both ap2 and pi (Figure 9D), trichomes than the organs of the outer whorl. Four of 104
grown at 25°C, have an outer whorl of four cauline leaf- of these organs scored had ridges of the type that cover
like organs topped with stigmatic papillae, as in the ap2 anther Iocules, three of 104 had stigmatic tissue at their
single mutant, but with an increased tendency toward apex. These organs develop on the time course of petals.
carpellody. The second whorl is variable, in both organ The third whorl primordia develop into stamens, although
number and organ identity. There are one to four organs, frequently capped by a stigma. The lateral stamens are
with an average of 2.5 (54 organs in 22 flowers scored). often missing. These flowers are self-fertile, indicating
When there are four organs, they occupy the positions normal pollen development and dehiscence of at least
normally occupied by petals in wild-type flowers. When some of the anthers.
there are fewer organs, their positions are irregular. The
identity of these organs varies from cauline leaf to solitary
carpel, with most being intermediate and having charac- DISCUSSION
teristics of both leaf and carpel. These organs show no
staminody, in contrast to the ap2 phenotype. Frequently
these second whorl organs are fused along one margin Our reason for analyzing these homeotic mutations is to
with the gynoecium, as shown in Figure 10. Mixed second understand the processes that allow cells in flowers to
whorl organs are mosaics of leaf and carpel, with a tran- recognize their appropriate developmental fate. Similar
sition zone one to five cells wide between the typical studies of developmental mutations in Drosophila have
epidermal cell types of these organs visible at the bound- revealed many of the strategies by which cellular identity
aries between different types of tissue. There are no third is established in early insect embryos (Lewis, 1978; Akam,
whorl organs, as in pi single mutants, and the gynoecium 1987; Scott and Carroll, 1987).
is similar to that in pi homozygotes. It seems that all of the genes described here act in
At 16°C, ap2 pi double mutant flowers consist of leaf- allowing cells to recognize their position in the developing
like organs, sometimes topped by stigmatic tissue, in the flower. AP2 and PI may also be required for the appearance
positions normally occupied by sepals and petals. These of organ primordia in some whorls. None of the mutations
surround a gynoecium like that of pi single mutants. Sec- has any regular effect other than elimination of organs, or
ond whorl organs are only occasionally missing (3 missing converting their fate. Beyond this general appraisal, we
out of 28 flowers), and can possess ovules (20 organs out can only describe the functions of the products of the
 Downloaded from https://academic.oup.com/plcell/article/1/1/37/5970194 by guest on 30 January 2023

Figure 9. Phenotypes of Double Mutant Combinations Grown at 25°C.

(A) ag ap3.
(B) ag pi.
(C) ag ap2.
(D) ap2 pi.
(E) ap2 ap3.
Bar = 1 mm.
 Flower Development in Arabidopsis 49

ap2 ap3 double mutants, every organ exhibits carpellody.

Other examples of Arabidopsis mutations whose pheno-
types include free carpels are known (Robbelen, 1965;
Haughn and Somerville, 1988). Even in wild-type plants,
the final flowers to develop can exhibit extreme carpellody.
agamous is an exception to this: either singly or in a double
mutant combination, ag has not been observed to have
any organ with carpelloid characteristics. Perhaps the wild-
type product of this gene is required for any cell to differ-
entiate to a type specific to carpels.
A comparable conclusion might be drawn regarding PI,
since flowers homozygous for pi-1 alone or with other

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mutations never have cells differentiating in a manner
characteristic of staminal cells. The wild-type PI product
may be required for any cell to differentiate to a stamen-
specific fate.
The nearly additive interactions observed between ag
and each of the other mutations suggests an absence of
interaction of the AG gene product and the products of
the other genes. In contrast, double mutant combinations
involving ap2 and either ap3 or pi display phenotypes that
are not observed in the single mutants, suggesting direct
or indirect interaction at some level. The nature of these
interactions precludes establishment of epistatic relation-
ships between the genes: for example, the second whorl
organs of ap2 ap3 can be leaflike or carpelloid, indicating
Figure 10. Fusion of Second Whorl Organs to the Central Gyn- that neither gene is epistatic to the other.
oecium in an ap2 pi Flower.
It must be pointed out that only one allele of each of
Note the row of ovules present where the two organs are fused. these mutations has been described here, and the different
Also note the carpel-like tissue and leaflike tissue sectors in the phenotypes found at different temperatures for the tem-
second whorl organ on the left. perature-sensitive alleles ap2-7 and ap3-1 indicate that
Bar =100 Mm. many of the phenotypes seen in these mutants are due to
partial loss of function of the wild-type product. Consistent
with this, the phenotype of a newly isolated mutant allele
flower development genes in the most general terms. The of ap2 (designated ap2-2; D.R. Smyth, J.L. Bowman, and
AP2 product, for example, must be involved in the process E.M. Meyerowitz, work in progress) is much more abnor-
by which the organs of the first and second whorls interpret mal than ap2-7. At 25°C, its flowers usually have only two
their position, and it acts at the time when the primordia outer whorl organs that are carpelloid, no second or third
of these organs are first forming. This product could, whorl organs, and a relatively normal gynoecium. When in
therefore, be a part of a signal from some region of the heterozygous state with ap2-7, an intermediate phenotype
plant or flower to these whorls, part of the receptor for results. Another three mutations with phenotypes between
such a signal, or part of the machinery of the cell that acts these extremes, flo2, flo3, and flo4 (Haughn and Somer-
subsequently to stimulation of the receptor. In the absence ville, 1988), have recently been shown to be allelic with
of knowledge of the cell types in which the AP2 product ap2-7 (L. Kunst, J. Martinez-Zapater, and G.W. Haughn,
acts, we cannot differentiate between these general hy- personal communication). One important task for the fu-
potheses. Identification of the cell type in which the gene ture is to obtain a wider allelic series for each of these
product acts could be obtained either by mosaic analysis genes.
or by using molecular cloning of the gene to identify and It has been suggested that communication between
locate the gene product. developing organs of adjacent whorls leads to sequential
One principle suggested by the phenotypes of the plants specification of the fate of the primordia in each whorl
described is that carpel fate may be a ground state, and (Heslop-Harrison, 1963; McHughen, 1980; Green, 1988).
that the wild-type products of these genes act to alter this However, it cannot be that each whorl depends on the
ground state to allow other organs to differentiate. Car- proper differentiation of the adjacent and outer one, since
pellody is the most prevalent phenotype among these there are examples in the results reported here of incorrect
mutations: ap3 makes the third whorl carpelloid, ap2 specification of each whorl, with correct specification of
causes carpellody of the first whorl, and in the ap2 pi and the adjacent inner whorl. For example, ap2-7 homozygotes
50 The Plant Cell

at 16°C have leaves instead of sepals, but nearly normal Exceptions to this are those cells on the borders between
petals; ap2-1 plants that at 29°C have staminoid organs mosaic patches, which may be intermediate in morphol-
instead of petals have a normal third whorl of stamens; ogy, and organs in the second whorl of ap2-1 flowers.
ap3-1 plants at 25°C or 29°C have carpels in the third A notable feature of the development of most of the
whorl, but a normal gynoecium. That inner organs specify abnormal organs is that they develop on a time course
the adjacent outer whorl cannot be simply true, either. characteristic of their whorl and not of their organ identity.
Another class of model that is better supported by the With one exception, the various organs that develop in
evidence is that the flower primordium is divided into fields whorl 2 develop later than the adjacent whorl 3 organs;
or compartments, each consisting of adjacent whorls. The this is true even when all of the organs of both whorls are
early-acting gene AP2 may specify a developmental state of the same type (as, for example, in ap2-1 at 29°C). The
for the cells that will later give rise to whorls 1 and 2, identity of the organ to which a primordium develops, and
whereas the wild-type AG gene may specify a different

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the time course of its development, are thus separable.
state for those that will become whorls 3 and 4. Similarly, The only exceptions to this are the petals that form in
the PI product may set aside a separate fate for those whorl 3 of agamous flowers, which develop in parallel with
cells that will give rise to whorls 2 and 3. Thus, the the second whorl petals.
combined action of all of these genes is the delineation of Finally, it should be noted that the mutations described
concentric ring-shaped compartments, each with a differ- here resemble similar, perhaps homologous, mutations in
ent fate. Even if something like this does occur, the present other species of plants, agamous is one typical sort of
information is insufficient to exclude other classes of double flower (Masters, 1869; Reynolds and Tampion,
models or to allow any speculation on biochemical mech- 1983); similar phenotypes were described in Matthiola
anisms. One thing is clear: there are few, if any, restrictions more than 400 years ago (Dodoens, 1568: see Saunders,
on the ultimate fate of the cells in any whorl. For example, 1921). Other genera in which mutants giving this pheno-
the organs of the second whorl can be leaves, sepals, type are known include Cheiranthus (Masters, 1869), Ar-
petals, stamens, or carpels, and those of the third whorl abis (Bateson, 1913), Petunia (Sink, 1973), and many
can be sepals, petals, stamens, or carpels, all as a result others. A similar, perhaps allelic Arabidopsis mutation,
of the manipulation of only a small number of the many multipetala, has been described as well (Conrad, 1971). A
genes that must be involved in specifying these organs. Capsella mutant with a phenotype quite similar to ap2-1
One question raised by any model requiring communi- was described as long ago as 1821. More recent descrip-
cation between adjacent developing regions is whether tions of this mutant are given by Dahlgren (1919) and Shull
the hormones known to act in plants are involved. The fact (1929). ap3 analogs have been reported in Cheiranthus
that a carpelloid stamen mutation in tomato can be re- (Nelson, 1929) and in Primula (Brieger, 1935); many other
verted to wild-type by application of gibberellic acid carpelloid stamen strains have been described (Meyer,
(GA3, Sawhney and Greyson, 1973) emphasizes the im- 1966), as have strains like ap3 or pi with conversion of
portance of this question. Two lines of evidence indicate petals to sepals (see Renner, 1959). The numerous reports
that the known hormones are not involved in the pheno- of mutants resembling those described in this paper indi-
types of the mutations described here. The first is that cate that the processes of floral development in Arabidop-
application of exogenous gibberellic acid (GA4+7 or GA3), sis are unlikely to be fundamentally different from those in
indole acetic acid, and kinetin had no effect on any of the any other plants.
mutants described (J,L. Bowman and E.M. Meyerowitz,
unpublished data). The second is that there are Arabidop-
sis mutants known that either fail to produce, or fail to
METHODS
respond properly to gibberellins, auxins, abscisic acid, and
ethylene; none of these mutations give phenotypes involv-
ing homeotic conversions (Koornneef et al., 1985; Bleecker The alleles studied, agamous-1,apetala2-1, apetala3-1, and pis-
et al., 1988; King, 1988). tillata-1 are in the Landsberg ecotype and homozygous for the
The fact that mosaic organs are composed of distinct erecta mutation. They were obtained from Maarten Koornneef
regions, with the epidermal cells in each region resembling (Departmentof Genetics, Wageningen Agricultural University,The
those normally found in a single organ, may indicate that Netherlands). Genetic nomenclature used here is based on rec-
individual cells in organ primordia make autonomous and ommendations of the Third International Arabidopsis Meeting
heritable decisions as to their fate at a time when the (East Lansing, Michigan, 1987). Wild-type alleles are symbolized
in block capitals and italics; mutant alleles in lower case italics.
primordium consists of only a few cells, and then multiply
Individualmutant allelesare designated by a number that follows
to form a clone of cells whose differentiation reflects the the mutant symbol and a hyphen (e.g., ap2-1, ap2-2). If not
choice made by their common ancestor. Also, most indi- specified, it is assumed that the mutant alleleis number 1. Doubly
vidual epidermal cells in the mutants differentiate into cell mutant stains were constructed by manualcross-pollination,using
types normally found in wild-type flowers, thus showing as parents strains homozygous for individual mutations. The
normal cellular differentiation but in inappropriate places. resulting F1 plants were allowed to self-pollinate, and double
 Flower Development in Arabidopsis 51

mutants were selected from the F2 plants. To establish strains thaliana (L.) Heynh. mit ver&ndertem Bletenbau und Bleten-
involving agarnous-l, which is sterile when homozygous, hetero- stand. Biol. Zentralbl. 90, 137-144.
zygotes were used as initial parents. Seeds were planted on a
peat moss/potting soil/sand (3:3:1, v:v:v) mixture in 55-mm pots. Dahlgren, K.V.O. (1919). Erblichkeitsversuche mit einer dekan-
The plants were grown in incubators under constant cool-white drischen Capsella bursa pastoris (L.). Svensk Bot. Tidskriff 13,
fluorescent light at 16°C, 25°C, or 29°C, and 70% relative 48-60.
humidity. Green, P.B. (1988). A theory for inflorescence development and
For scanning electron microscopy (SEM), young, primary inflo- flower formation based on morphological and biophysical analy-
rescences were fixed in 3% glutaraldehyde in 0.025 M sodium sis in Echeveria. Planta (Bed.) 175, 153-169.
phosphate (pH 7.0) at 4°C overnight, and then transferred to 1% Haughn, G.W., and Somerville, C.R. (1988). Genetic control of
osmium tetroxide in 0.05 M sodium cacodylate buffer (pH 7.0) at morphogenesis in Arabidopsis. Dev. Genet. 9, 73-89.
4°C for 12 to 24 hr. They were then rinsed in 0.025 M sodium Heslop-Harrison, J. (1963). Sex expression in flowering plants.
phosphate (pH 7.0) and dehydrated in a graded ethanol series at In Meristems and Differentiation, 16th Brookhaven Symposium

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4°C. This material was critical point dried in liquid carbon dioxide. in Biology, (Upton, NY: Brookhaven National Laboratory), pp.
Individual flowers were removed from infiorescences and mounted 109-125.
on SEM stubs. Organs were dissected from individual flowers by
King, P.J. (1988). Plant hormone mutants. Trends Genet. 4, 157-
applying pressure with glass needles. The mounted specimens
162.
were coated with gold and palladium (4:1) in a Technics Hummer
V sputter coater after each dissection. SEM was performed on Koornneef, M., Cone, J.W., Karssen, C.M., Kendrick, R.E., van
an ETEC Autoscan scanning electron microscope at an acceler- der Veen, J.H., and Zeevaart, J.A.V. (1985). Plant hormone
ating voltage of 20 kV, and the images were photographed on and photoreceptor mutants in Arabiclopsis and tomato. UCLA
Kodak 4127 film. Symp. Mol. Ceil. Biol. New Ser. 35, 103-114.
Koornneef, M., de Bruine, J.H., and Goettsch, P. (1980). A
provisional map of chromosome 4 of Arabidopsis. Arabidopsis
Inf. Serv. 17, 11-18.
ACKNOWLEDGMENTS Koomneef, M., van Eden, J., Hanhart, C.J., Stam, P., Braaksma,
F.J., and Feenstra, W.J. (1983). Linkage map of Arabidopsis
thaliana. J. Hered. 74, 265-272.
This work was supported by grant PCM-8703439 from the Na-
tional Science Foundation (to E.M.M.). J.L.B. is supported by
Lawrence, G.H.M. (1951). Taxonomy of Vascular Rants (New
York: Macmillan).
National Institutes of Health training grant 5T32-GM07616. D.R.S.
thanks colleagues at Monash University for supporting his sab- •Lewis, E.B. (1978). A gene complex controlling segmentation in
batical leave at Caltech. We thank our laboratory colleagues for Drosophila. Nature 276, 565-570.
discussions and constructive criticism, and P. Koen of the Caltech Masters, M.T. (1869). Vegetable Teratology: An Account of the
Electron Microscope Facility for advice. Principle Deviations from the Usual Construction of Plants (Lon-
don: Ray Society).
McHughen, A. (1980). The regulation of tobacco floral organ
initiation. Bot. Gaz. 141,389-395.
Received October 26, 1988. Meyer, V.G. (1966). Flower abnormalities. Bot. Rev. 32, 165-
195.
Mtiller, A. (1961). Zur Charakterisierung der Bitten und Infloresz-
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Scott, M.P., and Carroll, S.B. (1987). The segmentation and Recent observations have shown that stigmatic tissue, which was
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