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INTRODUCTION
Most angiosperm species are induced to flower in response
to environmental conditions such as day length and temper-
ature, and internal cues including age. Following germina-
tion, the shoot meristem produces a series of leaf meristems
on its flanks. However, once floral induction has occurred
flower meristems arise instead. These in turn produce a
determinate number of floral organ primordia, which
develop individually into sepals, petals, stamens or carpels.
Thus, flower formation can be thought of as a series of
distinct developmental steps - floral induction, the formation
of flower primordia and the production of floral organs.
Mutations disrupting each of the steps have been isolated in
a variety of species, suggesting that a genetic hierarchy
directs the flowering process (for review see Weigel and
Meyerowitz, 1993).
Recently, studies of two distantly related dicotyledons,
Arabidopsis thaliana and Antirrhinum majus, have shed
light on some of the molecular genetic mechanisms under-
lying the flowering process. For example, the specification
of floral organ identity depends on the products of at least
three classes of homeotic genes, acting alone and in combi-
nation (Bowman et al., 1989, 1991; Carpenter and Coen,
1990; Schwarz-Sommer et al., 1990). Several of these genes
are transcription factors whose conserved DNA-binding
domain has been named the MADS box (Schwarz-Sommer
et al., 1990; Coen and Meyerowitz, 1991).
Earlier-acting genes controlling the identity of floral
meristems have also been characterized. Flower meristems
are derived from the flanks of the indeterminate inflores-
cence meristem in both Arabidopsis and Antirrhinum. Two
of the factors that determine that these meristematic cells
will develop as flowers are known. In Arabidopsis, they are
the products of the LEAFY gene (Weigel et al., 1992) and
the APETALA1 gene (Mandel et al., 1992). When inacti-
vated by mutation, the flanking primordia develop into
structures combining the properties of flowers and inflores-
cence shoots in both leafy mutants (Schultz and Haughn,
1991; Huala and Sussex, 1992; Weigel et al., 1992) and, in
different ways, in apetala1 mutants (Irish and Sussex,
1990). In Antirrhinum, the homologue of the Arabidopsis
LEAFY gene is FLORICAULA (Coen et al., 1990) and that
of the APETALA1 gene is SQUAMOSA (Huijser et al.,
1992). The latter pair contain MADS box domains.
An allelic series of strong, intermediate and weak leafy
mutants has recently been characterized, presumably con-
taining progressively higher levels of LEAFY activity. This
721
Development 119, 721-743 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
Mutations in the APETALA1 gene disturb two phases of
flower development, flower meristem specification and
floral organ specification. These effects become manifest
as a partial conversion of flowers into inflorescence
shoots and a disruption of sepal and petal development.
We describe the changes in an allelic series of nine
apetala1 mutants and show that the two functions of
APETALA1 are separable. We have also studied the
interaction between APETALA1 and other floral genes
by examining the phenotypes of multiply mutant plants
and by in situ hybridization using probes for several
floral control genes. The results suggest that the products
of APETALA1 and another gene, LEAFY, are required
to ensure that primordia arising on the flanks of the
inflorescence apex adopt a floral fate, as opposed to
becoming an inflorescence shoot. APETALA1 and
LEAFY have distinct as well as overlapping functions
and they appear to reinforce each other’s action. CAU -
LIFLOWER is a newly discovered gene which positively
regulates both APETALA1 and LEAFY expression. All
functions of CAULIFLOWER are redundant with those
of APETALA1.APETALA2 also has an early function in
reinforcing the action of APETALA1 and LEAFY, espe-
cially if the activity of either is compromised by
mutation. After the identity of a flower primordium is
specified, APETALA1 interacts with APETALA2 in con-
trolling the development of the outer two whorls of floral
organs.
Key words: Arabidopsis, flower development, apetala1,
inflorescence, meristem, cauliflower
SUMMARY
Control of flower development in Arabidopsis thaliana by APETALA1and
interacting genes
John L. Bowman1, John Alvarez1, Detlef Weigel2, Elliot M. Meyerowitz2and David R. Smyth1,*
1Department of Genetics and Developmental Biology, Monash University, Clayton, Victoria 3168, Australia
2Division of Biology 156-29, California Institute of Technology, Pasadena, California 91125, USA
*Author for correspondence
722
series has been useful in deducing the wild-type LEAFY
function (Huala and Sussex, 1992; Weigel et al., 1992).
Further, the LEAFY gene product seems to interact with that
of APETALA1 as the phenotype of the apetala1 leafy double
mutant deviates more severely from wild type than do the
phenotypes of either single mutant (Huala and Sussex, 1992;
Weigel et al., 1992).
Here we describe the phenotypes of an allelic series of
nine apetala1 mutants and compare their morphology and
development. We have also examined interactions between
apetala1 mutants and those of five other floral control genes.
One of these is a new gene, CAULIFLOWER, which greatly
enhances the ap1 mutant phenotype when in homozygous
recessive form. The results extend those of an earlier study
of the apetala1-1 mutant (Irish and Sussex, 1990) and
provide insights into the various roles APETALA1 plays in
controlling the identity of flower primordia and of floral
organs that arise on them.
MATERIALS AND METHODS
Origin of mutant alleles
The origins of the nine mutant ap1 alleles studied are summarized
in Table 1.
The cauliflower-1 allele was identified as an enhancer of ap1-1
in the F2of a cross between an ap1-1 plant of Landsberg erecta
background and a wild-type Wassilewskija (Ws) ecotype plant
(Feldmann and Marks, 1987). Selfing of plants homozygous for
ap1-1 and heterozygous for cal-1 resulted in a segregation of 498
ap1-1 to 175 ap1-1 cal-1 plants in the next generation. The
enhanced phenotype was identified in five out of five F2families,
indicating that it is homozygous in the Ws background. Two lines
of evidence suggest that the apparently recessive cal-1 allele is
specific to Ws. Firstly, no enhancement of the ap1-1 mutant
phenotype was observed after crosses to different ecotypes
(Columbia, Niederzenz, Bensheim and Kiruna, a late flowering
ecotype). Secondly, mutant ap1 alleles isolated in four different
ecotypes, Landsberg erecta, Columbia, Nossen and either Estland
or Limburg (Table 1), display the non-enhanced phenotype. For
phenotypic analyses, the recessive cauliflower allele was back-
crossed into the Landsberg erecta ap1-1 background five times.
The cal locus is on chromosome 1 in the vicinity of ga4 and dis2.
Mutants of other genes used in this study have been described
previously (Table 2).
Genetic and phenotypic analyses
Plants were raised in a mixture of equal parts of perlite and a com-
mercial seed-raising mix and irrigated with nutrient solution.
Except where noted, phenotypic analyses were done on plants
grown in a greenhouse maintained at close to 25˚C under constant
illumination with fluorescent lights (cool white) at approximately
130 µE m−2sec−1in addition to natural daylight. For other tem-
peratures, phenotypic analyses were performed on plants grown
under continuous fluorescent lighting at 15±1˚C or 30±1˚C.
All double mutants segregated close to the expected ratio of 1:15
in F2families. For ap1-1 ap2-2,ap1-4 lfy-5 and ap1-5 ap2-1, seeds
were harvested from individual ap1 or ap2 F2plants, and the novel
double mutant phenotype was observed in a 1:3 ratio in the F3gen-
eration. The ap1-1 cal-1 lfy-6 and ap1-1 cal-1 tfl-2 triple mutants
were identified by harvesting seed from individual ap1-1 cal-1 F2
plants and observing the novel phenotype segregating 1:3 in the F3
J. L. Bowman and others
Table 1. Origin of mutant ap1 alleles
Allele Isolation no. Effect Ecotype Mutagen Origin
1−strong Landsberg er EMS* M. Koornneef
2 S1504 intermediate-strong Landsberg er EMS present study
3 S1350 weak Landsberg er EMS present study
4 S1735 intermediate Landsberg er EMS present study
5 S1732 weak Landsberg er EMS present study
6 La77313/2 intermediate Landsberg er not recorded P. Maher (ex M. Koornneef)
7 S2723 strong Columbia EMS present study
8 axillaris intermediate Estland or Limburg not recorded A. D. McKelvie (ex M. Koornneef)
9 LC500 strong Nossen EMS L. Comai
*ethylmethane sulfonate.
The ap1-1 and ap1-3 mutations are the result of splice site acceptor changes at the third and fifth introns, respectively, and ap1-2 is a glycine to aspartate
change at a conserved residue in the MADS box (Mandel et al., 1992).
Table 2. Properties of loci tested for interactions with ap1 mutations
Allele
Locus Summary of mutant phenotype used Effect Reference
leafy (lfy) flower meristems partially converted to inflorescence meristems 5 weak Weigel et al. (1992)
,, 6 strong ,,
cauliflower (cal) no effect alone, interacts with ap1 mutants to fully convert flower 1 ? present study
meristems to inflorescence meristems
terminal flower (tfl) apex of inflorescence meristems converted to floral meristems 2 strong Alvarez et al. (1992)
apetala2 (ap2) homeotic conversion of outer two whorls of floral organs
sepals converted to leaves, petals partially converted to stamens 1 weak Bowman et al. (1989)
sepals converted to carpels, petals mostly absent 2 strong Bowman et al. (1991)
sepals converted to carpels, petals mostly absent 9 intermediate ,,
agamous (ag) homeotic conversion of inner two whorls of floral organs
stamens converted to petals, carpels converted to another flower 1 strong Bowman et al. (1989)
,, 2 strong Yanofsky et al. (1990)
,, 3 strong Bowman et al. (1991)
723Flower development in Arabidopsis
generation. The heterozygous effect of cal-1 in the ap1-1 back-
ground was shown by crossing ap1-1 cal-1plants with ap1-1 plants
and observing a phenotype intermediate to that of the two parents
in the F1plants. The heterozygous effect of cal-1 in the ap1-5 and
ap1-1 tfl-2 backgrounds was shown similarly. The triple mutant
ap1-1 ap2-2 ag-1was identified as follows. A novel phenotype was
recorded in the F2of a cross between an ap1-1 plant and a plant
homozygous for both ap2-2 and pi-1 and heterozygous for ag-1.
Seeds were collected from individual F2ap1-1 plants. Some of the
F3families segregated the novel phenotype at the ratio of about
1:7. Each of these families homozygous for ap1-1 segregated ap2-
2and ag-1 but not necessarilypi-1. This segregation ratio (ap2 and
ag are linked and show about 25% recombination) and the segre-
gating phenotypes indicate that the novel phenotype corresponds
to the ap1-1 ap2-2 ag-1 triple mutant. That no different phenotype
was noted in those families also segregating pi-1 suggests that the
ap1-1 ap2-2 ag-1 pi-1 quadruple mutant is indistinguishable from
the ap1-1 ap2-2 ag-1 triple mutant.
For phenotypic analyses, usually the first fifteen structures
produced by the primary inflorescence meristem after cauline leaf
production were examined.
Scanning electron microscopy (SEM) was performed as
described in Alvarez et al. (1992) except that an accelerating
voltage of 20 kV was used and images were recorded on either
Kodak Plus-X Pan 220 professional film or Ilford FP4 film.
In situ hybridization
Fixation of tissue, sectioning, hybridization and washes were
carried out as described by Drews et al. (1991). Briefly, inflores-
cences were fixed for 3 hours in 3.7% formaldehyde, 5% acetic
acid and 50% ethanol. Fixed tissue was dehydrated with ethanol,
cleared with Histo-Clear (National Diagnostics) and embedded in
paraffin (Paraplast Plus). Embedded tissue was sliced into serial 8
µm sections with a Reichert-Jung 2040 microtome and attached to
microscope slides coated with poly-L-lysine (Sigma). [35S]UTP
probes were generated by run-off transcription with T3, T7 or SP6
RNA polymerase (Promega). Probes were synthesized from
templates described by Weigel et al. (1992) and Mandel et al.
(1992). In the case of AP1, probes were synthesized from a region
of the gene that did not cross-hybridize with any other cloned genes
(Mandel et al., 1992). Slides were dipped in nuclear emulsion,
exposed for from one to two weeks, developed for 2.5 minutes in
Kodak D19 and stained with toluidine blue.
RESULTS
The apical meristem of Arabidopsis thaliana generates
leaves until the plant becomes florally induced. Induction
occurs relatively rapidly under the influence of long days or
much later under short-day conditions (Napp-Zinn, 1985). It
results in the apical meristem generating the primary inflo-
rescence shoot and bractless flowers are now produced on
its flanks instead of leaves (Fig. 1). The leaves and flowers
arise in a continuous phyllotactic spiral, and the number of
flowers produced is indeterminate. The plant bolts soon after
the first flower primordia develop due to increased internode
elongation between several of the later-formed leaves
(cauline leaves) and between flowers. A cluster of earlier-
formed rosette leaves remains at the base of the stem. Each
of the cauline leaves contains an axillary inflorescence
meristem that reiterates the development of the primary
inflorescence. The inflorescence is a raceme with the devel-
opment of individual flowers proceeding acropetally (from
basal to apical positions; Müller, 1961).
Arabidopsis flowers are composed of four concentric
whorls of organs, with four sepals in the outermost whorl,
four petals in the second whorl, six stamens in the third
whorl (four medial and two lateral) and two fused carpels
occupying the center of the flower (Fig. 2B; Müller, 1961;
Hill and Lord, 1989; Smyth et al., 1990). Their early devel-
opment has been described in detail and divided into twelve
stages (Smyth et al., 1990; Fig. 2A).
Fig. 1. Schematic representation of a wild-type Arabidopsis
thaliana plant (left) and some of the alterations in apetala1
mutants (right). The roman numbers refer to the positions of the
cauline leaves and the arabic numbers refer to the positions of
flowers. Floral positions 1-5 are referred to as basal and positions
11 and above are referred to as apical. In all cases, the
inflorescences arising in the axils of cauline leaves (at I and II)
develop in the same manner as the main inflorescence. A-F
represent structures found in the basal flower positions of apetala1
mutants or in double and triple mutant combinations involving
apetala1 alleles. (A) Determinate branched structure, common in
strong apetala1 mutants. (B) Pedicellar inflorescence on a
determinate branched structure, present in strong apetala1
mutants and in apetala1 plants heterozygous for cauliflower-1. (C)
Second order inflorescence (what would normally be a flower
meristem behaves instead as an inflorescence meristem), present
in strong apetala1 mutants. (D) Second order inflorescence with a
third order inflorescence, present in strong apetala1 mutants. (E)
Positions normally occupied by floral meristems occupied by
inflorescence meristems; occurs in apetala1-1 cauliflower-1
plants. (F) Positions normally occupied by flowers occupied by
leafy shoots; occurs in apetala1 leafy-6 and apetala1 leafy-6
cauliflower-1 plants.
724 J. L. Bowman and others
Fig. 2. SEMs of wild-type (A,B) and apetala1 (C-I) inflorescences and flowers. Adaxial and abaxial refer to positions relative to the
inflorescence meristem, while lateral and medial refer to positions of floral organs relative to the floral meristem. Stages of flower
development are according to Smyth et al. (1990). (A) Vertical view of wild-type inflorescence meristem (im), with flower buds at stages
2-5 indicated. Note lateral (l) and medial (m) sepals arise at nearly the same level on the pedicel. (B) Nearly mature wild-type flower
(stage 12) partly dissected showing sepals (se), petals (p), stamens (st) and carpels (c). (C) Determinate branched structure characteristic
of the basal floral positions of ap1-1 inflorescences (Fig. 1A). In this case, the structure consists of five axillary flowers and a central
primary (p) flower. Note internode elongation between the lateral (l) and medial (m) first whorl organs of the primary flower. (D-E) Stage
6 ap1-1 flowers. The lateral (l) first whorl organs arise lower on the pedicel than the medial (m) first whorl organs. The axillary floral buds
(f) arise directly from the first whorl organs. Stipules (s) often flank the first whorl organs. The pattern of the second and third whorl organ
primordia is abnormal. (F) Developing ap1-1 inflorescence. The two basal positions normally occupied by flowers are occupied by second
order inflorescence meristems (1, 2). (G) A leaf-like primordium (arrowhead) flanked by stipules (s) and with an associated axillary
meristem is developing in a lateral position from the pedicel of this ap1-7 flower. (H) A much reduced axillary flower of an ap1-1/ap1-5
trans-heterozygous plant, consisting of only an abnormal, two-carpelled gynoecium. (I) An apical ap1-1 flower in which the first whorl
organs have failed to develop fully and the aborted primordia of the two lateral organs are visible (arrowheads). No second whorl organs
are present. Bar, 20 µm in A,D,E,G; 100 µm in B,F,H,I; 300 µm in C.
725Flower development in Arabidopsis
We have characterized nine recessive mutant ap1 alleles
(Table 1), one of which, ap1-1, has been described in detail
previously (Irish and Sussex, 1990). The mutant alleles can
be arranged into a phenotypic series (strong, intermediate
and weak) based both on the extent of inflorescence-like
character exhibited by ap1 flowers and the severity of alter-
ations in floral organ development. These two aspects will
be discussed separately.
ap1 mutations cause partial transformations of
flowers into inflorescences
The production of leaves is not altered in ap1 mutants.
However, those meristems that would give rise to a single
flower in wild type instead often give rise to a determinate,
branched structure comprised of several individual flowers
(Figs 1A, 2C). These structures consist of a central ‘primary’
flower with the extra flowers usually arising slightly later in
the axils of the first whorl organs of the primary flower. In
turn these secondary flowers may also have flowers arising
in the axils of their first whorl organs, and so on such that
tertiary and quaternary flowers may be formed. The overall
result is a complex branched structure. In some cases,
axillary flowers may not be subtended by a fully developed
organ (Fig. 2C). However, close inspection reveals that there
is usually an aborted organ primordium with flanking
stipules (Fig. 3K).
In ap1 mutant plants, the total number of flowers in the
branched structures varies acropetally (from bottom to top
of the inflorescence shoot) and with growth temperature
(Table 3). The most basal positions in relatively strong
mutants (alleles 1,9,7and 2) may have more than twenty
branched flowers arising from the position a single flower
would occupy in wild type, while the more apical positions
may be occupied by a single flower. Similarly, growth at
lower temperatures (15˚C) results in more flowers per
branch while growth at 30˚C results in less. In flowers of
intermediate ap1 alleles (4,6and 8), the determinate struc-
tures are seldom composed of more than eight individual
flowers, while in weak ap1 alleles (5and 3) no axillary
flowers were observed at 25˚C (Table 3).
Ectopic inflorescences may sometimes develop directly
on the pedicels of a p 1 flowers. They arise below the fir s t
whorl organs of the primary flowers of the determinate
branched structures (Fig. 1B). The ectopic structures are
usually not associated with any subtending organ or
aborted primordia, although occasionally they arise from
the axil of a cauline leaf-like organ. In strong alleles, these
‘pedicellar inflorescences’ are relatively common. For
example, 39 were observed on 20 a p 1 - 1 plants and 18 on
6 a p 1 - 7 p l a n t s .
In stronger alleles the main ‘flower’ meristem itself may
sometimes behave as a full inflorescence meristem,
producing an indeterminate number of flowers in a phyl-
lotactic spiral (Figs 1C, 2F). We term this a second order
inflorescence meristem (the first order is the normal apical
inflorescence meristem). Even third order inflorescence
meristems (Fig. 1D) may sometimes develop. These higher
order apices develop in a manner that is indistinguishable
from that of the apical inflorescence meristem (Fig. 2F), sug-
gesting that the transformation of flower to inflorescence
meristem is complete. Such conversions are almost always
restricted to the most basal positions of the main inflores-
cence stem. At 25˚C, there are only one or two per plant (if
any) for strong and intermediate alleles, and none at all for
weak alleles (Table 3). However, the number varies
markedly with temperature. For example, ap1-1 plants
grown at 15˚C had an average of 11 secondary inflores-
cences in the first 15 ‘flower’ positions, while at 30˚C only
one was seen on 15 ap1-1 plants (Table 3).
Thus strong and intermediate mutant ap1 alleles show
partial, and occasionally complete, conversions of flowers
to inflorescences, especially at basal positions.
Table 3. Mean number of flowers* per pedicel/peduncle† in ap1 plants
Allele:
strong intermediate weak
1 9 7 2 6 4 8 5 3 wild
15˚C 25˚C 30˚C type
flowers 1-5‡ ND 9.1 2.7 4.3 4.4 4.6 3.3 3.6 1.8 1.0 1.0 1.0
flowers 6-10 ND 3.7 1.3 3.7 1.9 2.8 2.1 2.2 1.5 1.0 1.0 1.0
flowers 11-15 ND 2.1 ND 3.3 1.6 1.8 1.8 1.8 1.3 1.0 1.0 1.0
second order 210 5 1 24 1 12 1 1 0 0 0 0
inflorescences§
no. of plants scored 19 16 15 15 15 15 15 15 15 15 15 15
All plants were grown at 25˚C unless otherwise noted. ND means not determined.
*A flower in this sense is defined as a floral-like structure that is terminated by a gynoecium. For example, if a primary flower has a flower-like structure
in each of the axils of its four first whorl organs, the number of flowers per pedicel/peduncle would be five. The axillary flowers are often incomplete, and
are counted as flowers only when they have a terminal gynoecium.
†Since the determinate inflorescence-like structures are neither true flowers nor true inflorescences, the term pedicel/peduncle is used to denote the floral
stem for these structures.
‡Flowers 1-5 refer to the first five determinate structures (Fig. 1A) produced by the apical inflorescence meristem following cauline leaf production.
Flowers 6-10 and 11-15 refer to the corresponding more apical structures.
§This represents the total number of times a meristem that in wild-type would be a floral meristem, behaved instead as an indeterminate second order
inflorescence meristem (Fig. 1C). Pedicellar (Fig. 1B) and third and higher order inflorescence meristems (Fig. 1D,E) are not included. None of these
classes are included in counts of the mean number of flowers per pedicel/peduncle.
726
ap1 mutations alter the development of floral
organs
The presence, identity and position of the outer two whorls
of organs are usually altered in ap1 mutants (Figs 2, 3;
Tables 4, 5).
First whorl effects
Strong alleles. The first whorl organs display features not
found in either floral or vegetative organs of wild-type
plants. They have been referred to as bract-like since they
resemble the bracts that subtend flowers of many other
species (Irish and Sussex, 1990). The shape of the organs is
neither sepal-like nor leaf-like, being elongate and rather
pointed and lacking a defined petiole (Fig. 3A). They do
have some leaf-like properties including epidermal cells
resembling those of leaves, stipules and late senescence,
while sepal-like characteristics include elongate cells on
their outer surface and the absence of stellate trichomes.
Other first whorl positions of strong mutants are not
occupied by fully developed organs but aborted primordia,
often flanked by stipules, are frequently present (Fig. 2I,
Table 4). In the more acropetal flowers, organs often fail to
fully develop in all four first whorl positions (Fig. 2I).
Observations of developing flowers show that the four
first whorl primordia are usually produced in a whorled,
cruciform pattern (Fig. 3B). However, the pattern is often
slightly twisted and, unlike wild type, the lateral organ
primordia are usually initiated much lower on the recepta-
cle than the medial primordia. In addition, later internode
elongation between organs, especially those of the first
whorl, results in their marked spatial separation (Fig. 2C).
In the more acropetal flowers (positions 20 and higher,
Fig. 1) of strong alleles grown at 25˚C, the medial first whorl
organs develop carpelloid characteristics such as stigmatic
papillae at their tips and rudimentary ovules along their
margins. This is accentuated when a p 1 - 1 plants are grown at
30˚C where the medial first whorl organs develop as solitary
carpels. The lateral organs are less affected in each case.
Intermediate alleles. The first whorl organs of basal
flowers in ap1 alleles 2,4and 6more closely resemble
leaves (Table 4). They are triangular in shape, flanked by
stipules (Fig. 3K), and have stellate trichomes present on
J. L. Bowman and others
Table 4. First whorl organs of ap1 flowers
Allele:
strong intermediate weak wild
1 2 6 4 5 3 type
Flowers 1-5, medial positions
sepals 0 0 0 0 0 0 40
petaloid leaves 0 0 0 0 22 21 0
leaves 0 34 28 38 11 7 0
bracts 16 6 12 2 6 7 0
carpelloid bracts 0 0 0 0 1 2 0
staminoid bracts 0 0 0 0 0 2 0
filamentous organs 1 0 0 0 0 1 0
absent 23 0 0 0 0 0 0
Flowers 1-5, lateral positions
sepals 0 0 0 0 0 0 40
petaloid leaves 0 0 0 1 0 0 0
leaves 0 22 9 36 17 23 0
bracts 14 4 5 0 19 6 0
carpelloid bracts 0 0 0 0 0 0 0
staminoid bracts 0 1 0 0 0 0 0
filamentous organs 5 2 0 1 1 7 0
absent 21 11 26 2 3 4 0
Flowers 11-15, medial positions
sepals 0 0 0 0 0 0 40
petaloid leaves 0 0 0 0 0 6 0
leaves 0 0 0 0 0 0 0
bracts 23 37 31 40 0 3 0
carpelloid bracts 0 0 4 0 19 9 0
staminoid bracts 0 0 1 0 20 22 0
filamentous organs 1 2 0 0 0 0 0
absent 16 1 0 0 1 0 0
Flowers 11-15, lateral positions
sepals 0 0 0 0 0 0 40
petaloid leaves 0 0 0 0 0 0 0
leaves 0 0 0 0 0 0 0
bracts 7 12 10 24 21 24 0
carpelloid bracts 0 0 0 0 0 0 0
staminoid bracts 0 0 0 0 0 0 0
filamentous organs 6 1 0 2 2 3 0
absent 27 27 26 14 17 13 0
Four plants grown at 25˚C were scored for each mutant.
Fig. 3. SEMs of floral organs of strong, intermediate and weak
apetala1 mutants. (A,B,I) strong ap1-1 mutant; (J,K) intermediate
ap1-2 and ap1-4 mutants; (C-H,L,M) weak ap1-3 and ap1-5
mutants. (A) Bract-like (b) organ in the first whorl position of a
primary ap1-1 flower. A tertiary flower (arrow) can be seen in the
axil of the first whorl organ of the secondary flower. (B) Stage 5
ap1-1 flower. The four first whorl organs are in cruciform
phyllotaxy, but the lateral (l) first whorl organs are lower on the
pedicel than the medial (m) first whorl organs. The pattern of the
third whorl organ primordia is abnormal. (C) Mosaic first whorl
organ from an ap1-3 flower, consisting of distinct sectors of
petaloid (p) tissue and tissue that appears intermediate between
sepal and leaf-like (se) tissue. (D) Mature apical ap1-5 flower. A
carpelloid bract-like organ (b) is seen in the first whorl as well as
two petal-stamen mosaic organs (ps) in the second whorl. (E)
Stage 5 ap1-3 flower. The first whorl organs are narrow and,
unlike wild type, fail to enclose the bud. (F) Young ap1-3
inflorescence. (G) Petal (p) and stamen (st) mosaic organ from the
second whorl of an ap1-5 flower. (H) Close up of (F) showing the
distinct sectors of petal (above) and stamen (below) tissue. (I)
Nearly wild-type petals occasionally develop in second whorl
positions of ap1-1 flowers. (J) Stage 5 ap1-2 flower. The lateral (l)
first whorl organ is flanked by stipules and is lower on the pedicel
the medial (m) first whorl organs. The first whorl organs of basal
ap1-2 flowers are generally leaf-like. One of the visible second
whorl organ primordia (arrowhead) is much larger than the other
and will most likely also develop into a leaf-like organ. (K) ap1-4
flower. The axillary flower bud (f) is not subtended by a fully
developed lateral first whorl organ but rather an aborted
primordium (l) flanked by stipules (s). A second whorl position is
occupied by a filamentous structure (arrowhead). (L) Apical ap1-5
flower at stage 5. One of the two visible second whorl primordia
(2) is close to the same level as the lateral third whorl primordium
(3). Thus the distinction between second and third whorl positions
is sometimes ambiguous. (M) Petal-stamen mosaic organ in an
ap1-5 flower. In this case, it appears that a lateral third whorl
primordium may have congenitally fused with a second whorl
primordium, resulting in the development of a hybrid organ with
petaloid (p) and staminoid (st) tissue. Second (2) and third (3)
whorl stamens are also visible. Bar, 20 µm in B,E,F,H,J-L; 40 µm
in C; 100 µm in A,D,G,I,M.
727Flower development in Arabidopsis
728
both adaxial and abaxial surfaces. They become more bract-
like acropetally (Table 4), and in flowers above about the
tenth to fifteenth position, they usually resemble those of
basal flowers of strong mutants.
Weak alleles. There is a marked difference between the
morphology of the medial and lateral first whorl organs in
ap1-5 and ap1-3. On basal flowers the medial organs super-
ficially resemble wild-type sepals but are usually mosaics of
central phylloid tissue and marginal petaloid tissue (Fig.
3C). The boundaries between the two types of tissue are
usually sharp. On more apical flowers, the medial organs
become leaf-like (flowers 6 to 10) and then bract-like
(flowers 11 to 15 and above, Table 4), although they are
often mosaics including sectors of bract-like, carpel and
stamen tissue (Fig. 3D). By contrast, the lateral first whorl
organs are more often bract-like, are not usually mosaics and
are often absent in the more apical positions (Table 4).
All first whorl organ primordia of weak alleles arise in a
cruciform pattern, but they are narrower and more pointed
than wild-type sepal primordia and the lateral primordia may
be lower on the receptacle compared to wild type (Figs 3E,
3F). There is little or no aberrant internode elongation
between the lateral and medial first whorl organs.
Second whorl effects
Strong alleles. The most common case for ap1-1,ap1-7 and
ap1-9 flowers is that no organs occupy the second whorl
positions (for ap1-1 data see Table 5; Irish and Sussex,
1990). This is due to the failure to initiate any second whorl
organ primordia (Fig. 2D,E). However, a small fraction of
the second whorl positions are occupied by either nearly
wild-type petals, morphologically wild-type stamens, or
mosaic organs with distinct sectors of petal and either
stamen or bract-like tissue. Although no petals were
observed in the sample of plants scored for Table 5, near-
wild-type petals are sometimes seen (e.g. Fig. 3I).
Intermediate alleles. Second whorl organs are often
absent, especially in apical positions. When present, their
structures differ markedly between the three intermediate
alleles scored (Table 5). For ap1-2, stamens and leaf-like
organs are often present, especially in basal flowers. The
leaves closely resemble those of the first whorl both in
mature morphology and in ontogeny. Their early growth is
rapid compared with normal second whorl petal primordia
(Fig. 3J). By contrast, ap1-4 plants often have petals and
petaloid leaves in early flowers, later changing to filamen-
tous organs (Fig. 3K; Table 5). In the second whorl of ap1-
6, petals and petaloid stamens predominate (Table 5).
When organs with properties of two organ types develop,
they may appear either as distinct mosaics or as blends.
Distinct sectors occur in organs composed of petal and
stamen tissue, whereas epidermal cells in petal/leaf organs
seem to combine the properties of petals (domed-shaped
with radial ridges) and leaves (rectangular with some
stomata present).
Weak alleles. In basal flowers, the second whorl is most
frequently occupied by morphologically wild-type petals as
in wild type (Table 5). However, in the more apical flowers,
such organs are often absent. When present, they are either
mosaic organs consisting of distinct stamen and petal tissue
(Fig. 3G,H) or morphologically wild-type stamens.
Third and fourth whorl effects
ap1 mutations have only minor effects on the third whorl.
Even in strong alleles there are often six stamens present.
However, the mean number per flower is slightly reduced
(5.2 in ap1-1 compared with 5.9 in wild type), and the
reduction is more marked in apical positions and at higher
temperatures (a mean of near 4 for ap1-1 plants raised at
30˚C) Observations on developing ap1 flowers show that the
boundary between the second and third whorl primordia is
J. L. Bowman and others
Table 5. Second whorl organs of ap1 flowers
Allele:
strong intermediate weak wild
1 2 6 4 5 3 type
Flowers 1-5
petals 0 0 21 39 65 54 80
petaloid stamens 4 0 19 0 10 6 0
stamens 0 15 3 0 0 1 0
filamentous organs 3 5 1 6 0 1 0
petaloid leaves 2 1 7 22 0 0 0
staminoid leaves 0 6 0 0 0 0 0
leaves 0 16 0 1 0 0 0
absent 71 37 29 12 5 18 0
Flowers 11-15
petals 0 0 0 6 1 14 80
petaloid stamens 0 2 19 1 10 16 0
stamens 1 10 7 2 15 16 0
filamentous organs 0 6 0 40 0 5 0
petaloid leaves 0 0 0 3 0 0 0
staminoid leaves 0 1 4 1 0 0 0
leaves 0 3 2 2 0 0 0
absent 79 58 48 25 54 29 0
Four plants grown at 25˚C were scored for each mutant.
Fig. 4. SEMs of apetala1 cauliflower inflorescences. (A) Wild-
type apex showing inflorescence meristem (im) and developing
stage 1-3 flower buds. (B) ap1-1 cal-1 inflorescence apex at a
similar age as that of A. Primordia produced by the apical
inflorescence meristem (im) are also behaving as inflorescence
meristems rather than flower meristems. (C) ap1-1 cal-1
inflorescence apex at a slightly older stage than B. Note the
second order inflorescence meristems (2) are morphologically
indistinguishable from that of the apical inflorescence meristem
(im). (D) Side view of a young ap1-1 cal-1 inflorescence. (E) Side
view of a much older ap1-1 cal-1 inflorescence. (F) Close up of
the structure occupying the most basal floral position in E. This
structure developed from a meristem (a second order inflorescence
meristem) that in wild type would have given rise to a single
flower. (G) Close up of the structure produced by a third order
inflorescence in F. Fourth (4) and higher order inflorescence
meristems are visible. (H) Apical inflorescence meristem (im) of
E with young second and third order inflorescence meristems
visible. (I) Differentiating ap1-1 cal-1 inflorescence. The flowers
resemble apical ap1-1 flowers. (J) ap1-1 cal-1 inflorescence apex
of a plant grown at 16˚C. Note the numerous developing cauline
leaves (cl) and cauline leaf primordia (arrowhead). (K) Flower
homozygous for ap1-1 and heterozygous for cal-1. A pedicellar
inflorescence (see Fig. 1B) is visible (arrowhead). (L) ap1-1 cal-1
tfl-2 inflorescence. No supernumerary inflorescence meristems are
evident and the apical inflorescence meristem terminates in a
flower (t). Bar, 20 µm in A,B; 40 µm in C,D,H; 100 µm in F,G,I,-
L; 500 µm in E.
729Flower development in Arabidopsis
730
often distorted (Fig. 3L), especially when fewer than six
third whorl primordia develop. In these cases, it was
sometimes difficult to assign an organ to the second or third
whorl. Relevant to this, second and third whorl organs are
occasionally fused (2 cases in 60 ap1-3 flowers; 1 case in
60 ap1-5 flowers, e.g. Fig. 3M), and mosaic organs consist-
ing of petal and stamen tissue may arise from intermediate
positions.
Finally, the fourth whorl gynoecium of ap1 flowers is
usually normal although occasionally the two carpels are not
completely fused, primarily in flowers arising higher on the
main stem.
It is worth pointing out that the structures of second and
higher order flowers within determinate branched flowers
(Fig. 1A) are at least as severely affected as the primary
flower and often more so. In the extreme, they may consist
of a solitary two-carpelled gynoecium (Fig. 2H).
In conclusion, study of floral organs in nine mutant ap1
alleles has revealed the following generalizations. (i) The
outer two whorls of organs are preferentially affected. (ii)
The lateral first whorl organs tend to be bract-like or absent.
(iii) The medial first whorl organs are highly variable and
can range from being bract-like to leaf-like to carpelloid. (iv)
Second whorl organs tend to be absent in strong mutants,
staminoid in intermediate mutants and petaloid in weak
mutants. (v) Mosaic organs tend to arise in the medial first
whorl and all second whorl positions, especially in the
weaker alleles. (vi) Later-produced apical flowers are more
severely affected than earlier produced basal flowers. (vii)
Higher temperatures result in a strengthening of the mutant
phenotype for each of the alleles.
cauliflower enhances the ap1 phenotype
An apparently recessive allele of the CAULIFLOWER
(CAL) locus, cal-1, significantly enhances the ap1
phenotype (see Materials and Methods for the origin of cal-
1). In ap1-1 cal-1 double mutants, each meristem that in
wild type would give rise to a single flower, consistently
behaves instead as an inflorescence meristem. These
meristems arise in a phyllotactic spiral on the flanks of the
main apex in positions where flowers would normally arise
(Fig. 4B). In turn these second order inflorescence
meristems immediately produce further meristems in a phyl-
lotactic spiral that also behave as inflorescence meristems,
i. e. third order inflorescence meristems (Figs 1E, 4B-D).
This process may be repeated several times resulting in the
production of fourth, fifth and higher order inflorescence
meristems (Fig. 4E-H). The transformed inflorescence
meristems are indistinguishable from the primary apical
inflorescence meristem.
After a large number of undifferentiated meristems has
developed, individual flowers may eventually differentiate
from a few of the last-formed meristems on any one branch
(Fig. 4I). They may arise from high order meristems on early
formed branches or from second or third order meristems on
later formed branches. The meristems develop into individ-
ual flowers that have a phenotype like that of apical ap1-1
flowers.
The number of orders of inflorescence meristem that are
produced varies acropetally and with environmental con-
ditions. When grown at 25˚C, the basal positions of ap1-1
cal-1 plants may have up to ninth order inflorescence
meristems, while the more apical positions may have only
second order inflorescence meristems. When grown under
unfavorable conditions (such as 30˚C) the basal positions
may have only second or third order inflorescence
meristems, whereas in the apical positions flowers develop
without any proliferation of higher order inflorescence
meristems. Perhaps the most striking phenotype occurs
when ap1-1 cal-1 plants are grown at 15˚C. In this case no
differentiation of flowers was observed after four months,
by which time twelfth order inflorescence meristems were
present. Additionally, such plants produce cauline leaves at
many positions within the second, third and higher order
inflorescence meristems (Fig. 4J).
ap1-1 plants heterozygous for cal-1 also display a slightly
enhanced mutant phenotype. Such plants have an increased
number of pedicellar inflorescences (Figs 1B, 4K; 87 pedi-
cellar inflorescences were scored on 11 plants grown at
25˚C). In addition, the frequency of second and third order
inflorescence meristems (Fig. 1D) developing in the basal
positions is increased. Thus the cal-1 allele is not completely
recessive, but weakly semi-dominant.
The cal-1 allele also modifies the phenotype of weak ap1
alleles. For example, the phenotype of ap1-5 cal-1 plants is
enhanced so that it now resembles that of the strongest
J. L. Bowman and others
Fig. 5. Expression patterns of APETALA1 in ap1 cal and tfl plants.
In situ hybridization of a AP1 anti-mRNA probe with longitudinal
sections (8 µm) through wild-type (A,B), ap1-1 cal-1 (C-F) and
tfl-2 (G-J) inflorescence apices. Each section was photographed in
two ways: bright field (A,C,E,G,I) and dark field (B,D,F,H,J).
(A,B) Wild type. A high level of AP1 signal is associated with
stage 1-2 flowers, as seen in the two stage 2 flowers (2), but no
signal is observed in the inflorescence meristem (im). From stage
3, signal becomes restricted to the cells that will give rise to the
outer two whorls (arrowheads on the stage 4 flower), whereas no
signal is seen in tissue that will give rise to whorls three and four.
Expression is maintained in the sepals (se) and petals (pe), at least
until the flower opens. In addition, a high signal is associated with
the pedicel (pd) throughout flower development. (C,D) Young
ap1-1 cal-1 inflorescence apex grown at 25˚C (similar to that in
Fig. 4C). A low but significant signal is seen in some of the
meristems (arrowhead) produced by the apical inflorescence
meristem (im). (E,F) Inflorescence of an older ap1-1 cal-1 plant
grown at 25˚C (similar to that in Fig. 4F). A level of AP1 signal
comparable to that of wild-type stage 1-2 flowers can be seen in
many of the supernumerary meristems (arrowheads). However, no
signal is detected in the older inflorescence meristems such as the
second order meristem indicated (2). In addition, signal is detected
in the pedicels/peduncles of the proliferating meristems (pd). This
signal appears stronger in the pedicels of the higher order
meristems. (G,H) Young tfl-2 inflorescence apex. Ectopic AP1
signal is present in the meristems (m) in the axils of the cauline
leaves (l). These meristems give rise to inflorescences in wild
type, but are transformed into flower meristems in tfl-2 plants. The
primary inflorescence meristem (im) also expresses AP1 around
its flanks (arrowheads), presumably in first whorl floral organ
primordia of a developing terminal flower. (I, J) Older tfl-2
inflorescence apex. AP1 signal can be seen in the first whorl sepal
of the developing terminal flower (se). In addition, ectopic AP1
signal is seen in the inflorescence stem (is) below the terminal
flower. This region acts as the pedicel of the terminal flower. Bars,
200 µm. Each autoradiograph is shown at the same magnification
as its corresponding bright-field micrograph.
731Flower development in Arabidopsis
732
mutant, ap1-1. Such double mutants have pedicellar and
second order inflorescence meristems in their basal
positions, structures that are absent in ap1-5 alone (Table 3).
Also, the floral organs that develop in ap1-5 cal-1 flowers
resemble those of ap1-1 plants in number and type.
Plants homozygous for cal-1 alone closely resemble wild
type although the first position above the cauline leaves is
often occupied by an inflorescence or a determinate
branched structure, a phenomenon that occurs in wild type,
but at a much lower frequency. Thus a major effect of cal-
1is only seen when it is combined with ap1 mutants.
Expression pattern of AP1 in cauliflower plants
Since AP1 expression is associated with floral meristems but
not inflorescence meristems (Mandel et al., 1992), we
examined its expression patterns in ap1-1 cal-1 plants at
different ages and grown under different environmental con-
ditions.
In wild-type flowers, AP1 mRNA is first detected in stage
1 flowers and is uniformly expressed at a high level through-
out stage 1 and stage 2 flower primordia (Fig. 5A,B; Mandel
et al., 1992). In stage 3-4 flowers, AP1 mRNA becomes
restricted to the first and second whorls of the wild-type Ara -
bidopsis flower, with no expression detectable in the pre-
sumptive third and fourth whorls. Expression is maintained
until past stage 12, throughout developing sepals and petals,
with no expression detectable in the stamens and carpels. In
addition, AP1 mRNA is present throughout the pedicel
during flower development but is not detectable in the inflo-
rescence meristem.
The expression of AP1 in ap1-1 mutant flowers occurs at
normal levels in stage 1-2 flowers (data not shown). In
contrast, its expression in ap1-1 cal-1 is markedly different.
A very low level of AP1 mRNA is detected in young ap1-
1 cal-1 inflorescences (Fig. 5C,D). However, in older inflo-
rescences a level of AP1 expression similar to that seen in
stage 1-2 of wild-type flowers is observed in some of the
higher order meristems (Fig. 5E,F). AP1 expression appears
to occur in the higher order meristems first, regions associ-
ated with the later formation of flowers but prior to any mor-
phological evidence of flower development. Also there is an
acropetal effect. In branches arising higher up the main
stem, AP1 mRNA accumulates in meristems that have been
through only a few orders of branching. In addition, a range
of AP1 expression is observed in the peduncles and pedicels
of the supernumerary meristems, with low expression in
those carrying low order meristems and increasing
expression in those of higher order meristems. These results
are all from plants grown at 25˚C. Very little AP1 expression
is observed in the supernumerary meristems of ap1-1 cal-1
plants grown at 16˚C (data not shown).
Thus the expression of A P 1 in ap1-1 cal-1 plants is
initially very low in primordia formed on the flanks of inflo-
rescence apices. However, depending on environmental con-
ditions, levels gradually increase in later formed primordia
until they reach near wild-type levels. At this stage the
primordia are apparently committed to develop as flo w e r s .
Interactions between ap1, cal and terminal flower
mutants
In terminal flower (tfl) mutant plants, the primary apical
inflorescence meristem becomes determinate and its growth
terminates in a flower composed of more than the normal
number of floral organs (Shannon and Meeks-Wagner,
1991; Alvarez et al., 1992). A few normal flowers may be
produced before termination. The inflorescence meristems
arising in the axils of the cauline leaves usually produce only
a single terminal flower (Alvarez et al., 1992). The tfl
phenotype has been interpreted as a conversion of the inflo-
rescence meristem into a flower meristem (Shannon and
Meeks-Wagner, 1991) or an invasion of the inflorescence
apex by flower primordia (Alvarez et al., 1992).
Since ectopic flower meristems are formed in tfl plants,
we examined the pattern of AP1 expression in these
meristems. The apical inflorescence meristem, which
develops into a terminal flower in tfl-2 mutant plants,
exhibits ectopic AP1 expression early in its development
(Fig. 5G,H). Later expression is present in the outer floral
whorls (Fig. 5I,J). In addition, ectopic expression is
observed in the inflorescence stem itself, which, in tfl plants,
acts as the pedicel of the terminal flower. A high level of
ectopic AP1 expression is also detected very early in
meristems in the axils of cauline leaves which will develop
into individual flowers (Fig. 5G,H). Thus in tfl-2 plants AP1
expression is initiated at the time when the identity of
ectopic flowers is established
Since t fl- 2 and ap1-1 cal-1 mutant plants display
seemingly opposite effects in the interconversion of flo w e r
and inflorescence meristem identities, we constructed all
mutant combinations. Firstly, cal-1 tfl- 2 plants were appar-
ently indistinguishable from t fl- 2 singles in families segre-
gating for both, further evidence that c a l - 1 i n flu e n c e s
development only when a p 1 is also mutant. Secondly, a p 1 -
1 tfl- 2 doubles display an additive phenotype except that
the terminal flower on the main inflorescence apex has few
if any axillary flowers. Overall the phenotype is close to
that of ap1-1 tfl- 1 plants recorded by Shannon and Meeks-
Wagner (1991). Finally, the triple mutant, ap1-1 cal-1 tfl-
2, displays an unexpected phenotype. Its morphology is
indistinguishable from that of the ap1-1 tfl- 2 double mutant
(Fig. 4L), meaning that t fl is fully epistatic to c a l - 1 i n
a p 1 - 1 mutant background. Furthermore, even heterozy-
gosity for t fl- 2 reduces the enhancement by c a l - 1 of the
a p 1 - 1 mutant phenotype. In ap1-1 cal-1 plants heterozy-
gous for t fl- 2 a maximum of three rounds of meristem pro-
liferation is observed, in basal positions, before flo w e r s
d i f f e r e n t i a t e .
Interactions between ap1, cal and leafy mutants
Like ap1, mutations of the LEAFY (LFY) gene also result in
the partial transformation of flower meristems into inflores-
cence meristems (Schultz and Haughn, 1991; Weigel et al.,
1992; Huala and Sussex, 1992). The basal positions in
strong lfy mutants exhibit complete transformation of
flowers to inflorescence shoots, while the more apical
flowers are only partially converted (Fig. 6A). However, a
major difference from ap1 is that the basal transformed
inflorescences are usually subtended by a cauline leaf and
the upper ‘flowers’ by a bract in lfy mutants. Also, the organs
that arise in lfy ‘flowers’ are more leaf-like, are often
initiated in a spiral, leaf-like manner (interior to a cruciform
first whorl) and significant internode elongation may occur
J. L. Bowman and others
733Flower development in Arabidopsis
734
between the organs (Weigel et al., 1992). Finally, secondary
flowers are less frequent than in ap1 mutants.
The AP1 and LFY gene products apparently interact to
specify floral meristem identity. Evidence for this comes
from studies of double mutants. A significant enhancement
of the lfy phenotype occurs when strong or weak lfy alleles
were placed in double mutant combination with the strong
mutant ap1-1 (Weigel et al., 1992; Huala and Sussex, 1992).
We have found a similarly dramatic interaction between
the intermediate ap1-4 allele and the weak lfy-5 allele, indi-
cating that an interaction does not depend on a complete loss
of AP1 function. While petals and stamens are common in
flowers of ap1-4 (Table 5; Fig. 6F) and in apical flowers of
lfy-5 (Fig. 6D; Weigel et al., 1992), ap1-4 lfy-5 flowers
consist entirely of leaf-like, carpelloid leaf-like and carpel-
loid organs (Fig. 6G,H). The phyllotaxy of the organs is
often intermediate between spiral and whorled (Fig. 6G,
compare Fig. 6C and Fig. 6E). In addition, flowers now
develop in the axils of some of the leaf-like organs. The
axillary flowers are much reduced, often consisting of only
two or three carpels. The primary inflorescence meristem of
ap1-4 lfy-5 plants usually terminates in a mass of carpelloid
tissue as in strong lfy plants (Weigel et al., 1992).
To examine if lfy and cal interact when in mutant form in
the same way as do ap1 and cal, plants were bred that carried
both lfy-6 and cal-1. Strikingly, their phenotype could not
be distinguished from that of lfy-6 single mutants indicating
a major difference between AP1 and LFY in their relation-
ship with CAL. This was reinforced when the phenotype of
the triple mutant ap1-1 cal-1 lfy-6 was examined (Fig. 6B).
Such plants were indistinguishable from ap1-1 lfy-6 double
mutants (Weigel et al., 1992), showing that lfy-6 is fully
epistatic to cal-1 in an ap1-1 background.
Expression pattern of LEAFY in cauliflower plants
Because the ap1 cal mutant combination apparently results
in a prolonged loss of the ability to specify floral meristem
identity at 25˚C, we analyzed the expression of the LFY gene
product in ap1 cal inflorescences grown at this temperature.
In wild-type flowers, LFY mRNA is first detected at a low
level in cauline leaf primordia and then in the flanks of the
inflorescence meristem in the floral anlagen (precursor) cells
(Fig. 7A,B; Weigel et al., 1992). It is uniformly expressed
at a high level throughout stage 1 and stage 2 flower
primordia, but in flowers later than stage 4 LFY mRNA
becomes preferentially localized to particular differentiating
organs such as the carpels and stamen filaments. Unlike
AP1,LFY expression is not significant in floral pedicels.
Whereas early LFY expression is normal in ap1-1 single
mutants (Weigel et al., 1992), expression in ap1-1 cal-1
double mutants is, like AP1, drastically altered. Only low
levels of LFY transcript are observed in the basal-most
primordia produced by young ap1-1 cal-1 inflorescence
meristems (Fig. 7C,D). These levels range from those
observed in wild-type cauline leaf primordia to those in
floral anlagen. However, a level of LFY expression compa-
rable to that observed in stage 1-2 wild-type flowers is seen
in older ap1-1 cal-1 inflorescences (Fig. 7E,F). As in AP1,
there is a marked acropetal effect. The expression of LFY is
greatest in the higher order primordia, those from which
mature flowers are likely to soon develop (Fig. 7E,F).
Strikingly, as with AP1, very little LFY expression is
detected in any of the supernumerary meristems of ap1-1
cal-1 plants grown at 16˚C (Fig. 7G,H).
Interactions between ap1 and floral homeotic
mutants
Since mutations at the AP1 locus also affect floral organ
identity, we constructed combinations of various ap1
mutants with floral homeotic genes controlling floral organ
specification (Table 2; Bowman et al., 1989; 1991).
apetala1 apetala2 double mutants
Mutations in apetala2 (ap2), like ap1, affect the outer two
whorls. In flowers of strong ap2 mutants (Fig. 8A), the
medial first whorl positions are occupied by solitary carpels,
while the lateral positions either lack organs or are occupied
by leaf-like organs. No second whorl organs are present and
the number of third whorl stamens is reduced to an average
of less than one per flower (Bowman et al., 1991). Flowers
of weak ap2 mutants have a first whorl of leaf-like organs,
with occasional axillary flowers in the basal positions (10
occurrences in 20 flowers at 25˚C). Organs that are inter-
mediate between petals and stamens occupy the second
whorl (Fig. 8D; Bowman et al., 1989; 1991). The severity
of changes in organ identity of all ap2 alleles increases
acropetally and with environmental conditions (Bowman et
al., 1989; 1991) as in ap1 alleles.
In terms of organ identity, strong ap2 alleles are largely
epistatic to both strong and weak ap1 alleles. For example,
in ap1-1 ap2-2 flowers (Fig. 8B,C), the medial first whorl
organs, when present, are carpels and the lateral first whorl
positions are generally lacking fully developed organs,
although there may be aborted primordia in these positions.
There are no second and few third whorl organs. When
present third whorl organs are either stamens or carpelloid
J. L. Bowman and others
Fig. 7. Expression patterns of LEAFY in ap1 cal plants. In situ
hybridization of a LFY anti-mRNA probe with longitudinal
sections (8 µm) through wild-type (A, B) and ap1-1 cal-1 (C-H)
inflorescence apices. Each section was photographed in two ways:
bright field (A,C,E,G) and dark field (B,D,F,H). (A,B) Wild type.
A low level of LFY expression is first detected in floral anlagen (a)
on the flanks of the inflorescence meristem (im). In stage 1-2
flower primordia, a high level of LFY expression is observed
throughout the primordia, as seen in the two stage 2 flowers (2). In
older flowers, LFY expression is concentrated in certain
differentiating organs such as carpels (c). (C,D) Young ap1-1 cal-
1inflorescence apex grown at 25˚C (similar to that in Fig. 4C). A
low but significant signal is seen in some of the meristems
(arrowhead) produced by the apical inflorescence meristem (im).
(E,F) A second order branch of an older ap1-1 cal-1 inflorescence
grown at 25˚C (similar to that in Fig. 4F). LFY signal comparable
to that of wild-type stage 2 flowers is seen in the higher order
meristems (e.g. arrowheads), but little signal is observed in the
second order inflorescence meristem itself (2) or in the lower
order supernumerary meristems surrounding it.
(G,H) Inflorescence apex of an ap1-1 cal-1 plant grown at 16˚C
(similar to that in Fig. 4J). Little LFY signal is observed in any of
the proliferating meristems or any of the subtending leaves. A leaf
(l) and a second order inflorescence meristem (2) analogous to
that in E are indicated. Bars, 200 µm. Each autoradiograph is
shown at the same magnification as its corresponding bright-field
micrograph.
735Flower development in Arabidopsis
736
stamens, and the fourth whorl carpels often fail to fuse
properly and may also have sectors of stamen tissue. In the
more apical positions, the primary flower of ap1-1 ap2-2
double mutants consists of only a single gynoecium
composed of 2 to 4 carpels. This is also observed in ap2-2
single mutant flowers. Flowers of other double mutant com-
binations, ap1-5 ap2-2,ap1-1 ap2-9 and ap1-6 ap2-9, also
resemble those of the respective ap2 single mutants (results
not shown).
In contrast, a dramatic interaction is observed in double
mutant combinations of ap1 with the weak allele ap2-1. For
example, when ap1-5 (a weak allele) and ap2-1 are
combined, the resulting double mutant flower (Fig. 8E)
closely resembles that of the strong ap2-2 allele (Fig. 8A).
The medial first whorl organs are now most often carpels,
although in the single mutants these positions are occupied
by organs that are bract-like (ap1-5, Fig. 3D) or leaf-like
(ap2-1, Fig. 8D). No second whorl organs form, even in the
basal flowers. Their development (Fig. 8F) is also similar to
that of ap2-2 (Bowman et al., 1991). Much stronger mutant
phenotypes are also seen when ap2-1 is combined with ap1-
1or ap1-2 (results not shown).
Weak ap2 mutants also enhance the inflorescence
character of the branched floral structures of ap1-1 mutants.
In ap1-1 ap2-1 double mutants, determinate structures
composed of upwards of 50 flowers have been observed
developing from positions that in wild type would be a
single flower (Table 6; Irish and Sussex, 1990). In contrast,
in double mutant combinations involving strong ap2
mutants, such as ap1-1 ap2-2, this effect is reversed such
that the determinate branched structures are seldom
composed of more than ten flowers (Table 6). However in
this case the frequency of second order inflorescence
meristems is increased (Table 6).
apetala1 agamous double mutants
agamous (ag) mutants affect the identity of the third and
fourth whorl. Third whorl organs develop into petals instead
of stamens and cells that would normally give rise to a fourth
whorl gynoecium behave instead like another flower
meristem. Thus ag flowers are indeterminate and consist of
a large number of whorls of sepals and petals (Fig. 8G)
Flowers of double mutant plants carrying strong ap1 and
ag alleles such as ap1-1 and ag-1 often appear to consist of
an indeterminate number of whorls of bract-like organs with
flowers arising in the axils of some of the organs of the first
and internal whorls (Irish and Sussex, 1990). The bract-like
organs are occasionally carpelloid (about 1 occurrence per
plant). However, further inspection reveals that some of the
internal organs are often petaloid (Fig. 8K) and near wild-
type petals may be common (Fig. 8L).
The development of the first whorl of ap1-1 ag-1 flowers
resembles that of ap1-1 flowers, with bract-like organs and
axillary flowers occupying those positions (Fig. 8H,I). The
pattern of organ primordia interior to the first whorl, in the
region of the second and third whorls, is irregular (Fig.
8H,I). This developmental program may repeat itself several
times, similar to the pattern seen in ag-1 flowers. However,
unlike ag1, the floral meristem may soon revert to an inflo-
rescence meristem and produce floral meristems on its flanks
in a phyllotactic spiral. This is related to another feature of
the ap1-1 ag-1 phenotype, conversion of the flower
meristem to a second order inflorescence meristem (Fig.
1D). This occurs at a higher frequency in ap1-1 ag-1double
J. L. Bowman and others
Table 6. Mean number of flowers per pedicel/peduncle
in ap1 ap2 plants
Genotype
ap1-1 ap1-1 ap2-1 ap1-1 ap2-2
flowers 1-5 9.1 20.0 3.5
flowers 6-10 3.7 7.7 2.8
flowers 11-15 2.1 4.8 2.4
second order 5 2 42
inflorescences
no. of plants scored 16 8 13
All plants were grown at 25˚C.
See Table 3 for explanation.
Fig. 8. SEMs of apetala1 apetala2 (A-F) and apetala1 agamous
(G-L) flowers. (A) Mature ap2-2 flower. The medial first whorl
organs are solitary carpels (c), no second whorl organs are present
and three staminoid organs occupy the third whorl. (B) Mature
ap1-1 ap2-2 flower consisting of 5 solitary carpels and a stamen.
The secondary flower (arrowhead) composed of a two-carpelled
gynoecium is not subtended by any organ. (C) A developing ap1-
1 ap2-2 flower at around stage 8, showing an incompletely fused
gynoecium (g), a medial first whorl organ which is carpelloid (m)
and an aborted lateral first whorl organ (l). Also visible is a
younger stage 4 flower (4). The development of these double
mutant flowers resembles that of ap2-2 single mutants (Bowman
et al., 1991). (D) Mature ap2-1 flower consisting of an outer whorl
of leaf-like organs, a second whorl of three staminoid petals
(arrowheads), five third whorl stamens and a central gynoecium.
(E) Mature ap1-5 ap2-1 flower. The medial first whorl positions
are occupied by two carpels (c), of which the one on the right has
a staminoid sector, a phenotype frequently observed in strong ap2
alleles (Bowman et al., 1991). Three staminoid third whorl organs
surround a central gynoecium. The phenotype of ap1-5 ap2-1
flowers resembles that of ap2-2 flowers (A), rather than that of
ap1-5 (see Fig. 3D) or ap2-1 (D) flowers. (F) Vertical view of an
ap1-5 ap2-1 inflorescence. The inflorescence meristem (im), a
stage 4 and a stage 7 flower are indicated. In the stage 7 flower,
there is a reduction in floral organ number and the gynoecial
primordium is abnormally shaped. The development of these
weak double mutant flowers resembles that of intermediate to
strong ap2 mutants (Bowman et al., 1991). (G) Mature ag-1
flower consisting of an outer whorl of sepaloid organs and an
indeterminate number of internal whorls of petaloid and sepaloid
organs. ag-1 and ag-3 flowers are indistinguishable. (H) Early
developing ap1-1 ag-1 flower. An axillary flower bud (f) is visible
in the axil of a first whorl organ. The pattern of organ primordia
internal to the first whorl is irregular. (I) Later developing ap1-1
ag-1 flower. Four axillary floral buds (f) are visible. The pattern of
organ primordia produced by the floral meristem (m) is irregular.
(J) Mature ap1-7 ag-3 flower. Two prominent axillary flowers are
visible, as well as two more internal axillary floral buds
(arrowheads). The internal whorls consist of petaloid and bract-
like organs. (K) Close up of the abaxial surface of a petaloid organ
from an ap1-7 ag-3 flower. The epidermal cells exhibit a mixture
of petaloid properties (cobblestone-like cells) and phylloid
characteristics (such as stomata). (L) Close up of the adaxial
surface of a petaloid organ from an ap1-7 ag-3 flower. The
epidermal cells are nearly indistinguishable from those of wild-
type petals. Bar, 10 µm in L; 20 µm in C,F,H,I,K; 100 µm in
A,B,D,E,G,J.
737Flower development in Arabidopsis
738
mutants (26 occurrences in 16 plants) than in ap1-1 alone
(7 in 20 plants).
apetala1 apetala2 agamous triple mutants
Because AP1 seems to interact with both AP2 and AG, and
the latter also interact (Bowman et al., 1991; Drews et al.,
1991), we were interested to examine the phenotype when
all three were in mutant form. In ap1-1 ap2-2 ag-1 triply
mutant plants (all strong alleles) the striking observation is
that ‘flowers’ proliferate extensively, producing an indeter-
minate number of primordia which remain relatively undif-
ferentiated. Interspersed among them are leaf-like organs
which are occasionally carpelloid (Fig. 9A). No structure
recognizable as a floral organ was ever seen to differentiate,
apart from the slightly carpelloid leaf-like organs. On the
basis of the size, shape and pattern of initiation, the
primordia appear to be a combination of inflorescence
meristems, flower meristems and organ primordia (Fig. 9D-
I).This is seen more clearly in an analysis of developing
structures (Fig. 9B,C). The pattern in which the primordia
arise is sometimes spiral (as in inflorescence meristems),
sometimes whorled (as in flower meristems) and sometimes
intermediate (Fig. 9D-I). Those meristems that produce
whorled primordia initially generate a first whorl of bract-
like organs, each with an associated axillary meristem. It
then either produces another whorl of similar organs (Fig.
9H), reverts to producing meristems in a phyllotactic spiral
(i. e. it acts like an inflorescence meristem, Fig. 9E), or
behaves intermediately between these two extremes (Fig.
9I).
DISCUSSION
AP1 controls two stages of floral development
Our study of an allelic series of ap1 mutants demonstrate
that the APETALA1 gene is required early in the determina-
tion of flower meristem identity, and later during initiation
and development of floral organs. Disruption of the early
function results in the partial transformation of flowers into
inflorescence shoots. Disruption of the later function causes
failure of flower organ initiation and changes in floral organ
identity, predominantly in the outer two whorls. These two
effects are separable in ap1 mutants. Inflorescence proper-
ties are weaker in later formed flowers on a plant whereas
floral organ changes are stronger. Also the weakest mutant
alleles display only the late phenotype. The two-fold activity
of AP1, as inferred from genetic analysis, correlates well
with the biphasic expression pattern of AP1 RNA (Mandel
et al., 1992). We will discuss the two stages in turn.
AP1 interacts with LFY in controlling floral
meristem identity
Early effects of ap1 mutants result in flower meristems
taking on some of the properties of inflorescence meristems.
The stronger the mutant allele, the more often the ‘flowers’
grow in an indeterminate, inflorescence-like pattern, and the
more often secondary and higher order flowers arise in the
axils of the outer floral organs (Table 3). The strongest
mutant alleles studied here (alleles 1,7and 9) do not result
in a complete transformation. Even so it seems likely that
they result in full loss of AP1 function. They have closely
similar phenotypes as expected of null mutants. Also the
ap1-1 allele has been sequenced and it carries a splice site
acceptor mutation in the third exon (Mandel et al., 1992).
This would result in loss of part of the K box, a conserved
domain downstream of the MADS box with sequence sim-
ilarity to the coiled-coil domain of keratins (Ma et al., 1991).
While not necessarily producing an inactive product, the
ap1-1 mutation would seem likely to severely disrupt its
function (Yanofsky et al., 1990; Ma et al., 1991).
Much more complete flower-to-inflorescence transforma-
tions can be obtained if strong ap1 mutants are combined
with strong lfy mutants, which independently cause partial
flower-to-inflorescence conversions (Huala and Sussex
1992; Weigel et al., 1992). Even in combinations of the
intermediate ap1-4 mutant and the weak lfy-5 mutant, we
have found that flower-to-inflorescence transformations are
greatly enhanced. This suggests that the products of AP1 and
LFY normally interact to reinforce each others activity to
specify flower meristem identity. The other four genes
studied here also interact with ap1 and/or lfy when in mutant
form. However, none has a significant effect on the identity
of flower meristems when mutant alone. It seems that AP1
and LFY are the major players in the specification of floral
meristem identity and that the other genes have secondary
or partially redundant roles.
Basis of early actions of AP1 and LFY
Firstly, it appears that the initial activation of AP1 and LFY
is coincident with floral induction. In Arabidopsis, factors
that mediate floral induction seem to be promoted by long
days and higher temperatures (Napp-Zinn, 1985). Both AP1
and LFY appear to be responsive to (i.e. activated by) these
same factors. The evidence for this is as follows. In ap1
single mutants, where LFY is still functional (Weigel et al.,
1992), transformation of flower meristems to inflorescence
meristems is much more complete if flowering is held in
check by lower temperatures (Table 3) or short days (Huala
and Sussex, 1992). This suggests that LFY expression also
remains at relatively low levels under these conditions,
allowing prolonged, inflorescence-like growth. Likewise in
lfy mutants, where AP1 is still functional, lower tempera-
tures (Weigel et al., 1992) and short days (Huala and Sussex,
1992) result in more complete inflorescence-like transfor-
mations. Activation of the two genes by those factors that
mediate floral induction could also explain the fading of
inflorescence-like properties in later-arising flowers of both
ap1 and lfy mutants. This is expected if levels of the factors
controlling floral induction progressively increase as an
induced plant ages.
Secondly, it seems that a threshold level of a factor (or
factors) regulated by both AP1 and LFY must be exceeded
for the complete specification of floral meristem identity to
occur. It seems that the AP1 and LFY gene products act in
a partially redundant manner in ensuring that this threshold
is reached. If the activity of either gene is compromised by
mutational change, the other can work to a limited extent to
allow the threshold level to be obtained. The meristem then
takes on some of the properties of a flower, the particular
properties dependent on which downstream functions are
J. L. Bowman and others
739Flower development in Arabidopsis
lacking (see below). If the threshold level is not reached (as
in the strong double mutant), the meristem behaves as an
inflorescence meristem. This threshold proposal, combined
with the proposed progressive strengthening of the factors
promoting floral induction, also allows a relatively simple
explanation for the overlapping patterns of acropetal
Fig. 9. SEMs of apetala1 apetala2 agamous inflorescences and flowers. (A) Mature ap1-1 ap2-2 ag-1 flower. This structure consisting of
numerous undifferentiated primordia and some leaf-like organs all of which have developed from a single floral meristem. (B) Young
ap1-1 ap2-2 ag-1 inflorescence. Flowers labelled d, e and f are shown in detail below. (C) Close up of the center of B showing the
youngest flowers and the apical inflorescence meristem (im). (D-E) Close up of flowers labeled d and e, respectively, in B. Interior to four
axillary meristems in a somewhat cruciform phyllotaxy (arrowheads) the floral meristem (m) is producing primordia in a phyllotactic
spiral. The ‘flower’ in E is similar to that in D only at a younger developmental age. (F) Close up of ‘flower’ labelled f in B showing the
floral meristem (m). (G) ap1-1 ap2-2 ag-1 flower consisting of organs with axillary meristems and other primordia in addition to the floral
meristem (f). Other floral structures can be seen with both spiral and whorled phyllotaxy. (H) ap1-1 ap2-2 ag-1 flower showing cruciform
phyllotaxy and axillary meristems associated with both lateral (l) and medial (m) first whorl organs. (I) ap1-1 ap2-2 ag-1 flower with
phyllotaxy intermediate between spiral and whorled. Four first whorl organs are present, lateral (l) and medial (m), each with an
associated axillary meristem surrounding the floral meristem (f). Bar, 20 µm in C-I; 100 µm in A,B.
740
variation shown by the strong, intermediate and weak allelic
series of both ap1 and lfy mutants. The weaker the muta-
tional change, the sooner the threshold is reached and the
earlier any inflorescence-like properties are lost.
Thirdly, it seems likely that, once present, the wild-type
AP1 and LFY gene products mutually enhance each other’s
activity (Fig. 10). This is proposed to account for the con-
siderably greater disruption of phenotypes seen when weak
or intermediate ap1 and lfy mutants are combined. The
enhancement seems to be primarily at the level of activation
of target genes rather than through directly influencing each
other’s transcription because each is transcribed when the
other is in mutant form (see above). Such mutual enhance-
ment, in combination with the requirement that threshold
levels of activity be reached, provides a basis for the sharp
transition between the production of inflorescence
meristems and flower meristems seen in wild type, and the
gradual transition observed in ap1 and lfy mutants.
Finally, although A P 1 and L F Y act in combination to
specify the identity of the floral meristem, their early roles
are not equivalent. In l f y but not a p 1 mutations the main
apex continues to produce cauline leaves/bracts. It could
be that L F Y but not A P 1 has a role in suppressing bract
formation (Schultz and Haughn, 1991; Weigel et al., 1992).
This may depend on the low level of L F Y e x p r e s s i o n
observed in the anlagen of floral meristems even before
stage 1 (Figs 7, 10). Another difference between A P 1 a n d
L F Y functions lies in development of the stems of indi-
vidual flowers. A P 1 but not L F Y is expressed in develop-
ing pedicels (Figs 5, 7; Mandel et al., 1992; Weigel et al.,
1992). The phenotype of a p 1 mutants indicates that AP1
activity is needed to identify this tissue as floral stem, sup-
pressing its potential to develop ectopic floral and inflo-
rescence meristems (Fig. 1B) and preventing its
e l o n g a t i o n .
The role of CAULIFLOWER
Neither AP1 nor LFY is expressed significantly in the early
primordia of ap1-1 cal-1 double mutants (although both are
in ap1-1 singles). This formally suggests that the expression
of both genes is positively regulated, either directly or indi-
rectly, by the CAL gene product.
A simple scheme for CAL function is that, in combination
with AP1, it elevates the expression of LFY and AP1 in early
flower primordia (Fig. 10). LFY expression is greatly
reduced compared to wild type in the first primordia
produced by the main inflorescence meristem in ap1-1 cal-
1plants (Fig. 7C,D). Even so LFY seems to be at a high
enough level to suppress bract (cauline leaf) development
although not enough to allow any floral characters to
develop. This leads to the proliferation of multiple, undif-
ferentiating inflorescence meristems. However, LFY
activity seems to gradually accumulate in the higher order
meristems of ap1-1 cal-1 plants (Fig. 7E,F), eventually
reaching a level required for floral meristem specification.
Flowers then develop and mature. We have proposed above
that LFY expression is relatively low at low temperatures
because of the reduced strength of factors inducing floral
development. The phenotype of ap1-1 cal-1 plants raised at
15˚C is consistent with this. The level of LFY is not suffi-
cient any more to suppress bract formation consistently and,
J. L. Bowman and others
Fig. 10. Cartoon of how known gene products may interact in the
specification of floral meristem identity and floral organ identity.
A section through one half of a floral primordium is represented as
a set of boxes, with the regions representing each whorl and field
indicated at the top. The types of organs occupying the four
whorls in wild-type flowers are also indicated: Se, sepal; P, petal;
St, stamen; C, carpel (Bowman et al., 1991). Arrows represent
positive interactions and barred lines represent negative
interactions. It should be emphasized that these interactions may
be either at the level of a direct influence of each other’s
transcription or at the level of regulation of downstream target
genes. Genes in bold indicate more important activities; genes in
parentheses denote relatively low levels of activity. The four
lower diagrams represent successively later stages of flower
development as defined in Smyth et al. (1990). In the floral
anlagen, a low level of LFY activity is required to repress bract
formation. During stages 1 and 2, each of the four genes (AP2,
AP1, LFY and CAL) are active throughout the primordium. They
act in combination, with AP1 and LFY the major players, to
specify the identity of the floral meristem. During stages 3 and 4,
organ identity genes are expressed in the meristem in three over-
lapping fields. Field A is represented by AP1 and AP2 activities
and field A function requires their combined presence. Field C
function is associated with AG expression in the inner two whorls.
Field A and field C functions are mutually antagonistic because
the expression of AP1 is repressed by AG activity in the inner two
whorls (Mandel et al., 1992) and expression of AG is repressed by
AP2 in the outer two whorls (Drews et al., 1991). Field B genes
PI and AP3 also come on at this stage. AP1, AP2 and LFY
probably still positively interact although this is not shown. By the
time that all the floral organ primordia have formed (stages 5-7),
the three fields of gene activity are established and act to specify
organ identity.
741Flower development in Arabidopsis
apparently, it never reaches the threshold at which flowers
are specified, even in apical positions (Fig. 7G,H)
The expression of AP1 parallels that of LFY in ap1-1 cal-
1plants (Fig. 5), suggesting that AP1 is also positively
regulated by CAL. These proposals also account for the fact
that the ap1-1 cal-1 lfy-6 triple mutant plants are indistin-
guishable from ap1 lfy doubles. If both ap1 and lfy are
inactive, it is irrelevant whether or not CAL is present to
boost their activity.
It may be that the role of CAL is fully encompassed by
AP1, since cal-1 reveals a phenotype only in an ap1 mutant
background. The enhancement of the phenotype of weaker
ap1 alleles by cal-1 such that they now resemble stronger
ap1 alleles suggests that CAL acts truly redundantly with
AP1 and that the strong ap1-1 cal-1 phenotype is the con-
sequence of none of the AP1 targets being activated. Even
though they act redundantly, CAL can substitute for only a
proportion of AP1 functions. If it could replace them all, the
ap1 CAL phenotype would be wild type.
Mutants with phenotypes closely similar to that of ap1-1
cal-1 have been seen in other species. Most familiar is that
of Brassica oleracea var botrytis, the cultivated cauliflower
of our meal table (Sadik, 1962). In the same family as Ara -
bidopsis, the origin of its abnormal phenotype is apparently
different. Its genetic basis depends upon one semi-dominant
mutation rather than a combination of two mutations. The
flowers that may break out of the inflorescence of cultivated
cauliflowers are wild type and not ap1-like (Yarnell, 1956).
The anantha mutant of tomato also results in the prolifera-
tion of undifferentiated inflorescence primordia, but in this
case a single recessive gene is responsible (Helm, 1951).
Thus it seems a range of different genetic alterations can
independently lead to the threshold conferring floral identity
not being reached.
Hyper-induction in terminal flower mutants
Several lines of evidence suggest that the tfl mutant
phenotype arises because such plants are ‘over-induced’ to
flower. Firstly, tfl plants are early flowering as compared to
wild type (Shannon and Meeks-Wagner, 1991). Secondly,
the number of normal flowers produced before the apical
inflorescence meristem terminates in a floral meristem is
influenced by environmental conditions (Shannon and
Meeks-Wagner, 1991; Alvarez et al., 1992). The same non-
inductive environmental conditions that enhance the pheno-
types of ap1 and lfy mutants, short days and low tempera-
tures, reduce the severity of the tfl phenotype. Thirdly, tfl-2
ap1-1 cal-1 triple mutants look just like tfl-2 ap1-1 doubles.
That is, loss or reduction of CAL activity has no effect in tfl-
2 ap1-1 plants. This suggests that factors responsible for
floral induction are already at high enough levels in tfl ap1
mutant plants such that LFY expression reaches levels suf-
ficient to determine floral meristem identity without the
stimulus provided by CAL. Fourthly, in tfl-2 plants, both
AP1 (Fig. 5G-J) and LFY (Weigel et al., 1992) are ectopi-
cally expressed in the transformed terminal meristems. That
is, these genes are activated in inappropriate parts of the
inflorescence meristem perhaps in response to the very high
levels of floral induction factors in tfl plants. Finally,
terminal flowers can be produced by the normally indeter-
minate inflorescence meristem in Sinapis alba, another
member of the Brassicaceae, if plants are given extreme
inductive growth conditions (Bernier, 1992).
Double mutant combinations of tfl and ap1 (Results;
Shannon and Meeks-Wagner, 1991) and of tfl and lfy (D. W.
and J. A., unpublished results) are apparently close to
additive, suggesting that the TFL gene product does not
interact directly with either of these main players. It may be
that the normal TFL function is to inhibit one or several
factors that promote floral induction (Koornneef et al., 1991)
and that all the effects of tfl mutations are direct or indirect
consequences of this release from inhibition. Of course, it is
possible that TFL function may negatively regulate AP1 and
LFY more directly in the apex of the inflorescence meristem.
In the absence of TFL function, AP1 and LFY would now
have the potential to become activated ectopically in these
apices.
The early role of APETALA2
AP2 also appears to have a role in the specification of floral
meristem identity in addition to its later role in the specifi-
cation of floral organ identity (see below). This seems likely
even though flowers of ap2 single mutants reveal few if any
inflorescence-like properties (Bowman et al., 1989; 1991).
The evidence comes from double mutants involving ap2
alleles and either ap1 or lfy mutations. In each case, the
flowers have enhanced inflorescence properties suggesting
that the AP2 product positively interacts with both AP1 and
LFY (Fig. 10). For example, combination of the strong ap2-
2mutant with the strong ap1-1 mutant produces a larger
number of flower-to-inflorescence conversions (second
order inflorescences, Table 6). If the weak ap2-1 allele is
involved with ap1-1, the flowers produced are much more
branched than in either single mutant (Table 6; Irish and
Sussex, 1990). [The fact that there is much less branching
in flowers of the ap1-1 ap2-2 double (Table 6) may result
from ectopic expression of AGAMOUS (see below).] ap2
mutants also interact with lfy mutants in a parallel manner
(Huala and Sussex, 1992). Similar intensified changes have
been observed by us in weak ap2-1 lfy-5 doubles (unpub-
lished).
These effects can be accounted for if, in the absence of
either AP1 or LFY activity, AP2 activity is now required for
the floral identity threshold to be reached in primordia devel-
oping on the flanks of the inflorescence apex (Fig. 10).
Later roles of AP1 in floral organ development
Recent models of Arabidopsis flower development propose
that the identities of the four whorls of floral organs are
specified by the products of at least four floral homeotic
genes APETALA2,APETALA3,PISTILLATA and
AGAMOUS (Fig. 10; Bowman et al., 1991). The genes are
suggested to function alone and in combination in three
overlapping fields. Field A (whorls 1 and 2) is represented
by AP2, field B (whorls 2 and 3) is represented by AP3 and
PI, and field C (whorls 3 and 4) is represented by AG. In
addition, it is proposed that the A and C functions are
mutually antagonistic (Bowman et al., 1991; Fig. 10).
Mandel et al. (1992) found that, from late stage 3, AP1 is
expressed in regions of flower primordia corresponding to
field A and proposed that AP1 is involved in determining
development of the outer two whorls. Observations on our
742
allelic series of ap1 mutants support this, as disruption is
predominantly confined to sepal and petal development. It
seems likely that AP1 promotes or acts as a partner in the
activity of AP2, the other known field A gene, in specifying
the identity of outer whorl organs (Fig. 10). This is because
double mutant combinations involving weak alleles, such as
ap1-5 ap2-1, exhibit a much enhanced change in organ
identity toward that of strong ap2 mutant alleles (Fig. 8E).
Mandel et al. (1992) also suggested that AP1 expression
may be negatively regulated by AG in whorls 3 and 4 (Fig.
10). This was proposed because AP1 is not normally
expressed in the inner whorls once AG comes on, but it is if
ag is in mutant form. Further, this negative regulation of
AP1 by AG may extend to the outer two whorls in ap2
mutants where AG is ectopically expressed in whorls one
and two (Drews et al., 1991). Consistent with this, strong
ap2 alleles are epistatic to strong and weak ap1 alleles with
respect to disruptions to the identity of floral organs (Fig.
8B).
Proposed interactions between AP1,AP2 and AG from
stage 3 can be summarized thus: AP2 negatively regulates
AG in field A, AG negatively regulates AP1 in field C, and
both AP1 and AP2 are necessary for A function (Fig. 10).
Mutant phenotypes of the outer whorl organs can be
accounted for as follows. In ap2 mutants, AG comes on and
inactivates AP1 in field A leading to carpelloid first whorl
organs. By contrast, in ap1 mutants expression of AP2 and
AG is largely normal but A function is disrupted resulting
in bract-like organs developing in place of sepals. In ag
mutants AP1 and AP2 act as usual in field A and the outer
whorl organs are normal sepals. However, these genes are
also ectopically active in field C and stamen to petal con-
version is one result. AP1 thus appears to be, along with
AP2, a component of the A function that regulates organ
identity in whorls one and two. This follows both from its
action in specifying organ identity in these whorls, and from
the regulatory interaction between AP1 and the C function
gene AG.
One effect of ap1 mutations may be an ectopic expansion
of field C activity into areas of the flower primordium where
field A function is now compromised. This could account
for why stamens sometimes arise in positions normally
occupied by second whorl organs and why second whorl
organs themselves do not develop. The latter could be
related to ectopic AG expression and the role that AG
normally plays in suppressing growth in the center of the
determinant flower primordium (Bowman et al., 1991). Con-
sistent with this, petals may reappear in second whorl
positions of ap1 flowers when AG is also inactivated (as in
ap1 ag doubles (Fig. 8K,L)). Finally, petal-stamen mosaic
organs seen in the second whorl of ap1 mutants (Fig. 3G,H)
could develop from primordia that straddle the A/C
boundary.
The phenotype of ap1 ap2 ag triple mutants
An observation that needs to be accounted for is the inability
of ‘floral’ meristems in ap1-1 ap2-2 ag-1 triple mutants to
develop mature organs of any type. Indeed, such meristems
seem to revert frequently to inflorescence-like meristems.
This is perhaps unexpected given that the two earlier acting
genes which control flower meristem identity, AP1 and AP2,
do not lead to such a phenotype when in double mutant com-
bination. The additional inactivation of the later acting AG
seems to be responsible.
It seems that, in the triple ap1 ap2 ag mutant, the ectopic
flower that normally develops inside an ag flower frequently
loses its floral identity and reverts back to inflorescence-like
growth. When a primordium first develops on the flank of
the triply mutant inflorescence meristem, sufficient activi-
ties of those factors that specify floral meristem identity are
apparently present to allow it take on floral identity and to
develop first whorl primordia (stage 3). However, perhaps
these activities fall away in the central cells of the flower
primordium and they revert to inflorescence-like growth. A
similar explanation might apply in ap1-1 ag-1 double
mutants where we found that many flowers also revert to
inflorescence growth patterns. The fact that loss of AG
seems to be responsible for the dramatic reversionary
phenotype of the triple mutant suggests that AG plays a role
in maintaining floral identity. Additional evidence for this
role is that in ag single mutants secondary flowers occa-
sionally arise in the axils of the sepals and such flowers are
more common in ag ap2 double mutants (unpublished
observations).
AP1 function in Arabidopsis resembles SQUA
function in Antirrhinum
AP1 has a cognate homolog in Antirrhinum majus, the
SQUAMOSA gene (Huijser et al., 1992). AP1 and SQUA
share 68% identity in their predicted amino acid sequence
and their patterns of expression in flower development are
closely similar (Mandel et al., 1992). The corresponding
mutant phenotypes also share some features. There is an
incomplete conversion of flower meristems into inflores-
cence meristems in squa mutants, even in null mutants
(Huijser et al., 1992). The range of floral organ types in squa
mutant plants is also somewhat similar to that in ap1 mutants
(Huijser et al., 1992).
Flower development in squamosa mutants is also envi-
ronmentally sensitive so it is difficult to compare directly
the relative severity of squa and ap1 mutational effects.
However, the phenotypes reported by Huijser et al. (1992)
reflect more frequent and more complete conversions of
floral meristems to inflorescence meristems. Conversely, if
flowers arise in squa plants, they usually seem to be less
severely affected than in ap1. These differences indicate that
AP1 plays a less important role than SQUA early in estab-
lishing floral meristem identity, but a more important role
later in the developing flower.
It seems likely that a core of conserved regulatory genes
underpin the control of floral development across flowering
plants. Even though such genes seem to have retained their
basic roles, modification of their expression patterns and
their interactions may account for at least some of the great
diversity in inflorescence and flower structure that has arisen
since flowering plants first appeared.
Special thanks go to Marty Yanofsky for making the AP1 probe
available prior to publication and for constructive comments on the
manuscript. We also thank Tom Jack, Gerd Bossinger, Megan
Griffith and Alan Neale for helpful discussion of the manuscript.
We are indebted to Maarten Koornneef and Luca Comai for
supplying genetic material. Gunta Jaudzems provided excellent
J. L. Bowman and others
743Flower development in Arabidopsis
microscopy facilities. J. L. B. was supported by a Short Term Fel-
lowship from the Human Frontiers Science Program and an Aus-
tralian Research Council Postdoctoral Research Fellowship. J. A.
held a Monash Postgraduate Scholarship. D. W. was supported by
a EMBO Long Term Fellowship and a Senior Fellowship from the
American Cancer Society, California Division. This work was
supported by an Australian Research Council Grant A19131181 to
D. R. S. and a US Department of Energy, Division of Energy Bio-
sciences Grant DE-FG03-88ER13873 and a US National Science
Foundation Grant MCB-9204839 to E. M. M.
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(Accepted 26 July 1993)
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