The Plant Cell, Vol.

2, 755-767, August 1990 O 1990 American Society of Plant Physiologists

Early Flower Development in Arabídopsis

David R. Smyth,' John L. Bowman, and Elliot M. Meyerowitz*
Division of Biology 156-29, California lnstitute of Technology, Pasadena, California 91 125

The early development of the flower of Arabidopsis thaliana is described from initiation until the opening of the bud.
The morphogenesis, growth rate, and surface structure of floral organs were recorded in detail using scanning
electron microscopy. Flower development has been divided into 12 stages using a series of landmark events. Stage
1 begins with the initiation of a floral buttress on the flank of the apical meristem. Stage 2 commences when the
flower primordium becomes separate from the meristem. Sepal primordia then arise (stage 3) and grow to overlie
the primordium (stage 4). Peta1 and stamen primordia appear next (stage 5) and are soon enclosed by the sepals
(stage 6). During stage 6, petal primordia grow slowly, whereas stamen primordia enlarge more rapidly. Stage 7
begins when the medial stamens become stalked. These soon develop locules (stage 8). A long stage 9 then
commences with the petal primordia becoming stalked. During this stage all organs lengthen rapidly. This includes
the gynoecium, which commences growth as an open-ended tube during stage 6. When the petals reach the length
of the lateral stamens, stage 10 begins. Stigmatic papillae appear soon after (stage l l ) , and the petals rapidly
reach the height of the medial stamens (stage 12). This final stage ends when the 1-millimeter-long bud opens.
Under our growing conditions 1.9 buds were initiated per day on average, and they took 13.25 days to progress
through the 12 stages from initiation until opening.

INTRODUCTION

The genetic control of flower development in Arabidopsis seed from the fruit have already been characterized in
has been the focus of several recent studies including our detail (Müller, 1961). Changes in the apical meristem as it
own (Pruitt et al., 1987; Komaki et al., 1988; Bowman et moves from vegetative to floral growth also have been
al., 1989; Hill and Lord, 1989; Kunst et al., 1989; Okada described by several authors (Vaughan, 1955; Miksche
et al., 1989). In each case the effects of mutations that and Brown, 1965). However, a detailed study of the inter-
specifically disrupt flower morphogenesis have been char- vening early stages of flower development in A. thaliana
acterized in detail. It is argued that such mutations are has not been reported.
likely to occur in genes whose products control the regu- In this paper we describe the structure of Arabidopsis
latory decisions that underlie flower development. Ulti- buds by scanning electron microscopy from their first
mately, the cloning and molecular characterization of such appearance as a buttress on the apical meristem until they
genes are likely to allow their mechanism of action to be open as fully developed flowers. By examining the shape,
deduced (Meyerowitz et al., 1989). To understand better size, and surface features of developing floral organs, we
the aberrant development in mutant plants, we have char- have divided the continuous process into 12 stages de-
acterized wild-type development in detail. fined by landmark events. The duration of each stage has
The mature flower of Arabidopsis thaliana has a simple also been estimated. This analysis provides a series of
structure typical of the Brassicaceae. It has a calyx of four reference observations of importance for interpreting the
free sepals and a corolla of four petals, whose positions mechanisms of action of genes that control flower devel-
alternate with those of the sepals. There are four medial opment in this model species.
and two lateral stamens, the former longer than the latter.
The superior gynoecium has two carpels whose locules
are separated by a false septum. Ovules arise on the RESULTS
parietal placentae on each side of the septum.
The structure and development of the Arabidopsis
flower from the time of its opening to the release of mature Description of Developmental Events

' Permanentaddress: Departmentof Genetics and Developmental Flower initiation in Arabidopsis occurs continuously in an
Biology, Monash University, Clayton, Victoria 3168, Australia. indeterminate spiral at each floral apex. Thus, flowers can
* To whom correspondence should be addressed. be placed in order of age and developmental stage by their
 756 The Plant Cell

position on an inflorescence. In this study all flowers on appear a little later in stage 5 (Figure 3A). [This definition
the main apex of three plants in which the oldest flowers of the beginning of stage 5 is slightly earlier than that in
had just opened were analyzed in detail. One plant was our earlier paper (Bowman et al., 1989), where it was
22 days old, the other two were 24 days old. Observations defined as occurring when the lengthening medial sepals
were made directly on primordia and buds at the earliest overlapped.] Stage 5 ends and stage 6 begins when the
stages of development. At later stages the buds are en- bud is fully enclosed by both medial and lateral sepals.
closed by sepals. To reveal the organs developing under- The tip of the abaxial sepal overlies that of the adaxial,
neath, one medial and the two lateral sepals were dis- whereas the two shorter lateral sepals meet or overlap
sected from the bud. After detailed examination they were underneath (Figure 2A).
photographedfrom the side and the lengths of the remain-
ing lateral sepal; one or two of the petals, one or two long
Stages 6 to 12
stamens, a short stamen, and the gynoecium were re-
corded as the linear distance between the base and the
tip of the organ in each case. (In some buds further organs From the start of stage 6, sepals completely cover the
had to be removed in turn to reveal previously hidden bud. During stage 6, primordia of the four long stamens
organs.) Detailed measurements of buds from one of the bulge out and become distinct from the central dome of
three inflorescences are shown graphically in Figure 1. The cells (Figure 3B). The two lateral stamen primordia arise
overall appearance of another inflorescence before dissec- slightly lower on the dome and develop slightly later. The
tion is shown in Figure 2A. petal primordia increase in size somewhat, but are still
Based on observations and measurements from these relatively small. A rim around ithe central dome of the
inflorescences, we have divided the continuous process of flower primordium now begins to grow upward to produce
flower development from initiation until the bud opens into an oval, hollow tube that will be the gynoecium.
Stage 7 begins when the growing primordia of the long
12 stages, as shown in Table 1. A summary of information
stamens become stalked toward their base (Figure 3C).
on later stages has been added from the study of Müller
This region will give rise to the filament, the wider upper
(1961).
region to the anther. This occurs when the long stamen
primordia are around 25 pm to 30 pm long overall. Vertical
Stages 1 to 5 growth of the slotted gynoecial tube keeps pace with that

Flowers arise on the flank of the apical meristem, which is

a dome of cells about 45 pm in diameter at this time
(Figures 28 to 2D). Stage 1 begins when a floral buttress

-
1200-
appears. This increases in size by lateral outgrowth from
Lat Sepal

-
T
the apex. At the time it reaches a breadth of about 22 pm ......... ....... Peta1
to 25 pm, a groove separates it from the main apex. This 1O00 - Long Stamen
defines the beginning of stage 2. Growth of the hemispher- Short Stamen
ical primordium continues almost at a right angle to the h --_-o--- Gynoecium
main apex, which is itself lengthening and widening at the 5
v
800:
point of attachment of the bud. Stage 3 begins when sepal
primordia appear. By now the flower primordium is 30 pm I
to 35 pm in diameter and becoming stalked with an incip-
6 600-
Z
ient pedicel. Its growth is also becoming more vertical. The
abaxial sepal primordium arises first, followed soon after
Y
400-
by the adaxial and the two laterals. The sepal primordia
arise initially as ridges that lengthen and curve inward until
they begin to overlie the dome-shaped flower primordium. 200-
Again, the abaxial sepal is the first to do this, at which
time stage 4 begins. Pedicel elongation continues concur-
rent with an increase in the diameter of the developing bud BudNumber:l 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
to about 65 pm to 70 pm. BudStage: 6 6 7 8 8 8 9 9 9 9 10 11 11 11 12 12 12 13
Stage 5 begins with the appearance of stamen and petal
precursors (Figure 2C). Primordia of the four medial (long) Figure 1. Length of Flower Organs in Buds on the Main Inflo-
stamens are first seen as wide outgrowths on the central rescence Apex of a 22-Day-01d Plant.
dome of cells. Barely visible are the four petal primordia The youngest bud scored (bud 1) was at stage 6 and the oldest
that arise between the sepals and close to their base. The (bud 18) had reached stage 13. The stage reached by each of the
two lateral (short) stamens develop from primordia that other buds is indicated.
 Early Flower Development in Arabidopsis 757

Figure 2. Scanning Electron Micrographs of the Main Flowering Apex of Arabidopsis.

(A) Vertical view of the apex of a 24-day-old plant in which the oldest flower has reached stage 14. (The sepals and petals of this flower
have spread during preparation.) Bar = 1000 /urn.
(B) Lateral view of the youngest buds on an inflorescence after the older buds have been removed. The stage reached by each bud is
shown. The abaxial (Ab), adaxial (Ad), and lateral (L) sepal on the stage 3 bud are also indicated. Bar = 10 nm.
(C) and (D) Vertical views of two inflorescences in which buds are arising in the opposite spiral sense. In (C) younger buds appear in a
counter-clockwise direction from the next older bud; in (D) the direction is clockwise. The stage of each bud is indicated. Petal (P) and
long stamen (LS) primordia are just visible on the oldest, stage 5 buds. Bars = 10 ^m.
 758 The Plant Cell

Table 1. Summary of Stages of Flower Development in A. thaliana Listing the Landmark Events that Define the Beginning of Each
Stage and Its Approximate Duration
Age of Flower
Stage Landmark Event at Beginning of Stage Durationa(hr) at End of Stagea(days)
1 Flower buttress arises 24 1
2 Flower primordium forms 30 2.25
3 Sepal primordia arise 18 3
4 Sepals overlie flower meristem 18 3.75
5 Petal and stamen primordia arise 6 4
6 Sepals enclose bud 30 5.25
7 Long stamen primordia stalked at base 24 6.25
8 Locules appear in long stamens 24 7.25
9 Petal primordia stalked at base 60 9.75
10 Petals level with short stamens 12 10.25
11 Stigmatic papillae appear 30 11.5
12 Petals level with long stamens 42 13.25

13b Bud opens, petals visible, anthesis 6 0.5

14 Long anthers extend above stigma 18 1
15 Stigma extends above long anthers 24 2
16 Petals and sepals withering 12 2.5
17 All organs fall from green siliques 192 10.5
18 Siliques turn yellow 36 12
19 Valves separate from dry siliques up to 24 13
20 Seeds fall
~ ~ ~~ ~~ ~ ~~ ~~ ~~ ~ ~~~

aEstimatedto nearest 6 hr.

Results for stages 13 to 20 (after the flower opens) are summarized from Müller (1961)where they were named B3 to B10. Their timings
are given separately because a different strain was grown under different conditions from those used in the present study.

of the long stamens (Figure 3D). Petal primordia are now the long stamens, flowers move into the final bud stage,
hemispherical, although still relatively small (about 25 pm stage 12 (Figure 4D). [This corresponds to stage B2 of
in diameter) (Figure 3C).The beginning of stage 8 is defined Müller (1961).] During stage 12 petals continue to lengthen
by another landmark of stamen development, when anther relatively rapidly. The growth rate of lateral sepals contin-
locules are visible as convex protrusions on the inner ues to slacken while stamens and the gynoecium lengthen
(adaxial) surface of the long stamens (Figures 3E and 3F). in concert. The anthers have almost reached their mature
These stamens have a total length of 55 pm to 60 pm at length of 350 pm to 400 pm for both the long and short
this time, most of it made up by the developing anther. stamens. Lengthening of the filaments now accelerates
Locules also appear soon after in the short stamens. rapidly. The upper part of the gynoecium becomes differ-
Petal growth now accelerates, and when the peta1 pri- entiated into a short, 1OO-pm- to 120-pm-longstyle, which
mordia become stalked, stage 9 begins (Figure 4A). This is slightly constricted toward its base. A sharp boundary
stage involves a rapid lengthening of all organs, especially separates it from a cap of stigmatic papillae (Figure 4D).
the tongue-shaped petals (Figure 1). These increase in Bud stage 12 ends when the sepals open and stage 83
length fourfold to fivefold, from about 45 pm up to 200 (Müller, 1961) (here renamed 13) begins (Table 1). The
pm. The stamens also grow rapidly. By the end of stage petals can be seen between the sepals and continue to
9, the media1 stamens are around 300 pm long. Most of elongate rapidly. The stigma is already receptive at this
this growth occurs in the anther region, which still accounts stage and anthesis occurs. Stamen filaments extend even
for over 80% of their total length. The gynoecium continues faster, and the length of the long stamens outstrips that
to elongate as an open, oval tube with a somewhat tapered of the gynoecium at the beginning of stage 14 (Figure 2A).
apex. The growth of sepals keeps pace so that the bud
remains completely closed.
Stage 1O begins when the fast-growing petals reach the Mature Flower
top of the short stamens (Figure 46). Soon afterward,
stage 11 begins when the upper surface of the gynoecium The mature floral organs show a range of different surface
develops stigmatic papillae, although their outward growth morphologies. The outer sepal surface has many stomata
is limited at first to regions not in contact with the overlap- interspersedamong irregularly shaped epidermal cells (Fig-
ping sepals (Figure 4C). Once the petals reach the top of ure 5A). In addition, there are always severa1 characteris-
 Early Flower Development in Arabidopsis 759

tically elongated cells (up to about 300 pm long) extending buds had reached stage 3 (Figure 6A). At 16 days several
longitudinally. Probable progenitors of these cells can be of the oldest buds on the main axis of some plants had
first recognized at stage 8 when the lateral sepals are only developed beyond stage 5 (Figure 6B). Buds also arise on
about 150 pm long (Figure 3E). The dista1 edges of the secondary floral apices; these occur in the axils of all
mature sepals are bordered by smaller epidermal cells that cauline leaves on the main stem. In our 117 plants there
are pale green in the live plant (Figure 5A). The outer were usually two such cauline leaves per main stem (mean
surface of the sepal may also have an occasional un- = 2.05 & 0.04, range 1 to 3). These leaves have clearly
branched trichome (Figures 2A and 6C). These are lacking developed stipules at each side of their point of attachment
from the inner surface, as are stomata and the elongated to the main stem (Figures 68 and 6C). At 16 days these
cells. Both surfaces of the peta1 blade are covered with secondary apices were much less advanced than the main
specialized, dome-shaped cells whose surface is finely apex. The oldest buds, flanked in turn by secondary cauline
ridged in a radial pattern (Figure 5B). Petals lack stomata. leaves, had only reached stage 2 or 3.
The surface of the anthers consists of epidermal cells of At around 18 days, the main inflorescence begins to
uniform size (Figure 5C) that have already been formed by elongate rapidly as the plant starts to bolt (Figure 6C). At
bud stage 1O (Figure 48). The surface of the ovary in the this time between four and seven buds per plant were
mature flower is made up of vertical files of epidermal cells observed to be at stage 6 or beyond on the main apex.
(Figure 5D). On the short style the cells are larger and are The secondary branches of the main inflorescence con-
interspersed with stomata. The densely packed stigmatic tinue to develop, with buds now at stage 5. The cauline
papillae are 20 pm to 35 pm long when the bud opens at leaves that overlie these buds develop trichomes. Further
the end of stage 12. The development of the stigma is inflorescences arise in the axils of the rosette leaves. At
shown in Figures 5E to 5G. By the end of stage 9 there is 18 days these have only just begun to develop, visible
not yet any differentiation at the tip of the gynoecium, under a cover of their own cauline leaf primordia.
which still has a slotted opening to the interior (Figure 5E).
This opening is maintained through stage 10, although it
becomes smaller. In addition, the eventual disc shape of Duration of Stages 1 to 5
the stigma surface is becoming apparent (Figure 5F). Dur-
ing stage 11, stigmatic papillae come to cover the entire To estimate the relative numbers of each of the five early
stigmatic surface. The gynoecium closes over, and the stages on the main axis, counts were made by light and
short style begins to develop (Figure 5G). scanning electron microscopy on 20 plants, four sampled
Nectaries are present in mature flowers at the base of on each of days 18,20,22,24, and 26, as shown in Table
the stamens (Figures 5H to 5J). Lateral nectaries arise at 2. An average of 7.8 floral primordia were present per
the base of the short stamens toward the end of stage 9 plant. There was no apparent change in the proportions of
(Figure 5H). They first appear as an outgrowth several flower primordia in each of the five stages during this
cells thick. By stage 11 they have formed either a large period. Overall, stages 1 and 2 were present most often
ridge (Figure 51), a single dome, or two domes of cells, with an average of more than two primordia per plant.
usually with several stomata at the apex in each case Stages 3 and 4 were less frequent (1.40 and 1.35 per
(Figure 5J). One or the other of the lateral stamens was inflorescence), whereas there was only a 0.7 chance of
absent in one-quarter of the flowers we analyzed in detail seeing a stage-5 bud.
(12 out of 47). In these the lateral nectary occupying the The absolute times spent in these stages can be as-
empty space was usually larger and more dome shaped. sessed if an estimate of the rate of bud production is
The occurrence of media1 nectaries at the base of the long available. This was obtained by recording the numbers of
stamens was sporadic. If present, they were narrower flowers on 20 plants on seven consecutive days beginning
than the lateral nectaries and arose later. on day 25, as shown in Table 3. Because of the difficulty
in accurately scoring primordia at stages 1 to 5 on live
plants, only buds with closed sepals (stage 6 onward)
Commencement of Flower Development were recorded. Nevertheless, this allowed the number of
buds per day that passed the stage 516 boundary to be
Before flower development starts, the main shoot apex deduced (Table 3). This was relatively constant from days
produces a limited number of rosette leaves. In a sam- 26 to 29, averaging 1.9 buds per day. [It fel1 significantly
ple of 117 plants grown under our conditions, the mean over the last 2 days (days 30 and 31), and these results
number of rosette leaves was 7.25 (SE = 0.07, range = have not been used in the following calculations.]
5 to 9). Because an average of 1.9 buds per day move out of
To trace the change from vegetative to floral develop- stage 5 over days 25 to 29, it can be argued that this is
ment, a sample of plants was fixed every 2 days beginning also an estimate of the rate of movement of buds into
12 days after incubation at 25OC. Buds were first visible stage 1 over the earlier period of days 18 to 26 because
on 14-day plants, by which time some of the first-formed the number of buds in stages 1 to 5 is constant over this
760 The Plant Cell

Figure 3. Individual Buds at Stages 5 to 8 with Sepals Dissected Away To Reveal Inner Organs.
 Early Flower Development in Arabidopsis 761

period (Table 2). Under such circumstances, stages 1 to 5 150" to the earlier bud (Figures 2C and 2D). The direction
occupy a total of 4.1 days on average (i.e., 7.8 buds per a new bud may take can be counter-clockwise(Figure 2C)
plant arising at the rate of 1.9 buds per day). Rounded off or clockwise (Figure 2D) from the previous bud, as viewed
to the nearest 6 hr, stage 1 lasts for 24 hr, stage 2 for 30 from the top. This direction is maintained by later buds on
hr, stages 3 and 4 are 18 hr each, and stage 5 occupies an axis giving, respectively, a righthanded or a lefthanded
only 6 hr (Table 1). helix.
One hundred seventeen mature plants were scored for
Duration of Stages 6 to 12 the direction of spiralling of their main axis. The numbers
were close to equality-60 spiralled clockwise, 57 counter-
The data in Table 3 also allow the total time in stages 6 to clockwise. Other axes arise on each plant as secondary
12 to be estimated. The number of buds moving into stage and tertiary branches of the main stem and as rosette
6 was in overall balance with the number moving out of inflorescences. These do not necessarily adopt the same
stage 12 (into 13 and beyond). For days 25 to 29, the helical sense as the primary apex. Indeed, there is some
average number moving out was 1.7 buds per day, close tendency for them to spiral the other way. On the 60 plants
to the 1.9 buds per day estimated earlier as moving into whose main apex spiralled clockwise, 136 further apices
stage 6. Thus, the average flux of buds moving through spiralled clockwise but there were 183 spiralling counter-
this period is 1.8 buds per day. Given that there is an clockwise. On the 57 counter-clockwiseplants, equivalent
average of 16.4 buds per inflorescence in these stages figures were 120 counter-clockwise but 135 clockwise.
(Table 3), a bud takes 9.1 days on average to pass through Overall, 256 apices adopted the same spiral sense as their
stages 6 to 12. main apex, whereas 318 were different, a significant de-
Finally, an estimate of the times spent in each of stages parture from equality ( x2 = 6.70 with 1 df, P c 0.01).
6 to 12 depends on counts of the numbers of bud per
plant at each stage. This requires the removal of sepals DISCUSSION
and was done only for the three inflorescences used to
define stages. The number of buds at each stage for one
of these is given in Figure 1. (Note that there was an Overall, the pattern of development of the Arabidopsis
additional bud at stage 6 on this inflorescence that was flower is similar to that described for other species in the
too small to measure accurately.) In the second infloresc- family Brassicaceae, such as t h e wallflower Cheiranthus
ence there were two buds at stage 6, two at 7, two at 8, cheiri (Payer, 1857; Sattler, 1973) and oil seed rape Bras-
five at 9, one at 1O, two at 11, four at 12, and one at stage sica napus (Polowick and Sawhney, 1986), in spite of the
14. In the third inflorescence there were two buds at stage fact that Arabidopsis flowers are much smaller throughout
6, three at 7, one at 8, four at 9, one at 10, two at 11, all stages of development. For example, in B. napus, the
three at 12, and one at stage 14. Because of these limited early flower primordium is about 170 pm in diameter when
data, stages have been estimated only to the nearest 6 hr sepals first appear (30 pm to 35 pm in Arabidopsis), and
(Table 1). The longest stage is stage 9 which, with 4.3 the mature bud is 5 mm long (1 mm in Arabidopsis). It.will
buds per inflorescence on average, lasts 2.5 days. Stage be of interest to determine whether this is a consequence
1O is the shortest-about 12 hr. of differences in the number of cells, their size, or both.

Direction of lnflorescence Spiral Organogenesis

Buds arise acropetally on an apex, with each new bud There has been controversy about the relative time of
arising around the apex at an angle of between 130" to appearance of floral organs and their location in whorls in

Figure 3. Individual Buds at Stages 5 to 8 with Sepals Dissected Away To Reveal lnner Organs.
(A) A bud at stage 5 in which a media1 and lateral sepal have been removed. One of the small petal primordia (P) is indicated. The
primordia of the long stamens (LS) are larger than that of the short stamen (SS).
(B) Lateral view of a bud at stage 6 in which the sepals had fully enclosed the bud. The stamen primordia are now dome shaped, whereas
the petal primordia are still relatively small. The gynoecium (G) will arise from the central dome of cells.
(C) Media1 view of a bud at stage 7 showing that the long stamen primordia are now constricted toward their base (arrow). The petal
primordia (P) have become dome shaped.
(D) Vertical view of a stage-7 bud showing that the stamens do not yet show locule ridges on their adaxial surface. The gynoecium (G) is
growing vertically as a slotted tube.
(E) A bud at stage 8 in which the stamen primordia have increased markedly in size, especially in relation to the petal primordia.
(F) Vertical view of a stage-8 bud in which locules (arrows) are now clearly visible in the stamens.
Bars = 10 fim.
762 The Plant Cell

Figure 4. Lateral Views of Buds at Stages 9 to 12.

(A) A bud early in stage 9 showing that the petal primordia have become wider toward the top as they start growing rapidly.
(B) A bud in which the petals have just reached the height of the lateral (short) stamens, marking the beginning of stage 10. Buds more
than double in size during the lengthy stage 9 [c.f. (A)].
(C) Stigmatic papillae (arrow) appear on the top of the gynoecium at the start of stage 11.
(D) Stage 12 is the final stage before the bud opens. It commences when the petals reach the height of the long stamens.
Bars = 100 ^m.
 Early Flower Development in Arabidopsis 763

the Brassicaceae flower. Whether all four sepals occupy observed by Miksche and Brown (1965) on Estland plants
one whorl or the two laterals occur in a separate whorl in soil-less culture. We estimated that an average of 1.9
outside that of the abaxial and adaxial sepals has been in buds arose per day on the main apex of 20 plants in this
question. The order in which sepals arise does not help study. The only other report from Arabidopsis is for three
settle the matter. It is clear in all species examined that plants of race Dijon grown at 20°C that produced buds at
the abaxial sepal is the first to appear. In Arabidopsis we a slightly faster rate-2.3 buds per day (Müller, 1961). It
agree with Hill and Lord (1989) that the lateral sepals arise is likely that these rates are influenced to a large degree
at around the same time as the adaxial sepal, although by environmental factors.
they are reported to arise earlier in Cheiranthus (Payer, The presence of stipules at the base of young cauline
1857; Sattler, 1973) and 6. napus (Polowick and Sawhney, leaves in Arabidopsis (Figures 66 and 6C) is of interest.
1986). What is established is that the laterals arise lower Payer (1857) was unable to see stipules in any species of
on the floral primordium (e.g., Figure 2B), and that their the Brassicaceae, but Arber (1931b) reported well-devel-
vasculature in the mature flower detaches below that of oped stipules on young leaves of Nasturtium officinale. We
the media1 sepals (Arber, 1931a). have reportedearlier that the presence of stipules on outer-
Petals arise in one whorl in the Brassicaceae flower. whorl organs of apetala2-7 flowers was evidence of their
Because their early development is relatively slow, there leaf-like structure (Bowman et al., 1989). Arber (1931a,
has been some doubt about whether they arise before or 1931b) also commented on the presence of filamentous
after the stamens. The petals have been reported to arise outgrowths at the base of some leaves and pedicels in a
before the stamens in C. cheiri (Payer, 1857; Sattler, 1973) range of Brassicaceae species. She called these “squa-
and after the stamens in 6. napus (Polowick and Sawhney, mules,” and distinguished them from stipules by their
1986). In Arabidopsis we could not confidently separate smaller diameter and their presence at the base of pedicels
their origin in time from that of the long stamens (see also as well as leaves. We were unable to see such organs on
Hill and Lord, 1989). any leaves or buds of wild-type Arabidopsis, although we
In A. thaliana and B. napus (Polowick and Sawhney, have seen them in various mutants, including apetala7.
1986) the four inner stamens arise a little earlier than the They may well be the “filamentous sepals” that Kunst et
two outer ones, whereas in Cheiranthus the order report- al. (1989) record between the outer floral organs of
edly is reversed (Payer, 1857; Sattler, 1973). In Arabidop- apetala2-7 mutants and that Komaki et al. (1988) report in
sis we found that one of the outer stamens was absent in mutant FI-54.
about one-quarter of the flowers examined. This has also The direction of flower production on any one main apex
been reported in three other races in which many flowers of Arabidopsis is apparently decided at random and is
had only four stamens (Müller, 1961). The pattern of maintained unchanged. In this regard it resembles the
gynoecium development seems to be similar in Arabidopsis pattern of phyllotaxis in many species including, for ex-
(the present study; Hill and Lord, 1989; Okada et al., 1989), ample, Pharbitis nil (Imai, 1927), Nicotiana tabacum, and
Cheiranthus (Payer, 1857; Sattler, 1973), and 6. napus N. rustica (Allard, 1946). It does seem in A. thaliana that
(Polowick and Sawhney, 1986). The major difference lies the spiral direction taken by secondary and tertiary
in the stigma, which is markedly bilobed only in the latter branches is somewhat influenced by the spiral pattern of
two species. the primary stem. We do not know whether this is the
Finally, our observations on nectaries, which concur with case in other plant species.
those of Müller (1961), show that they are variable in
presence, size, and shape. They arise late in flower devel-
Conclusion
opment (stage 9), and it may be that their growth is limited
by available space and nutrients.
Our definition of 12 stages in early flower development in
Arabidopsis will be useful in interpretingthe action of genes
Origin of Flowers that control this process. Already we have indirect evi-
dente that the product of the APETAL42 gene is active
The apical meristem of Arabidopsis changes from slightly during stages 2 to 4 (Bowman et al., 1989). Sepals, which
convex to distinctly dome shaped during the transition are affected by the apetala2-7 mutation, arise during this
from vegetative to floral growth (Vaughan, 1955; Miksche time. In the case of APETALA3, the gene product is appar-
and Brown, 1965). Flower primordia then arise on the flank ently active later when petals and stamens, the organs
of the apical meristem by periclinal divisions in cells be- affected by mutations in this gene, are differentiating(Bow-
neath the two-layered tunica. Under continuous light at man et al., 1989). Thepistillata mutation also affects petals
25”C, stage-3 buds were observed here after 14 days’ and stamens, and it is clear that in this case “petal”
incubation. Thus, the first bud is likely to have arisen at primordia arise as normal, but they differentiatewith sepal-
around 12 days in our plants, severa1 days later than that like properties under a petal timetable (Bowman et al.,
764 The Plant Cell

Figure 5. Surface Morphology of Mature and Developing Floral Organs.

(A) Outer surface of a mature sepal (left) showing several long epidermal cells (arrow), stomata, and a fringe of smaller cells. The lower
part of the adjacent petal (right) shows a regular transition from longer cells in the claw to more ovoid cells in the blade. Bar =100 Mm.
(B) Higher magnification of the ridged cells that cover the surface of the blade of mature petals. Bar =10 Mm.
(C) A mature stamen after dehiscence showing epidermal cells of uniform size and pollen grains. Bar = 100 Mm.
 Early Flower Development in Arabidopsis 765

Figure 6. Appearance of Immature Inflorescences on Plants at the Commencement of Flowering.

Rosette and cauline leaves have been removed.

(A) Vertical view of the main apex of a 14-day-old plant showing that the oldest bud has advanced to stage 3. Bar = 10 /im.
(B) A 16-day-old-plant with four buds beyond stage 5 (sepals closed) on the main apex. Two cauline leaves have been removed from the
main stem although their stipules remain (arrows). Secondary apices within these cauline leaves have also initiated flower development.
These apices are flanked in turn by younger cauline leaves, and their oldest flower primordium is only at stage 2. Bar = 100 /jm.
(C) Lateral view of an 18-day-old plant just beginning to bolt. The two secondary apices visible have advanced in concert, with buds
reaching stage 5 on each. Their cauline leaves show developing trichomes and stipules (arrows). A new apex is just visible in the axis of
one of the dissected rosette leaves (lower left) although flower development is not yet detectable under its cauline leaf primordia. Bar =
100 Mm.

Figure 5. (continued).
(D) The upper parts of a gynoecium in a stage-12 bud showing stigmatic papillae (SP), large cells and stomata on the short style (ST),
and smaller epidermal cells on the surface of the ovaries (OV). Bar =100 ^m.
(E) to (G) Structure of the developing stigma. The surface of the gynoecium is smooth and slotted at the end of stage 9 (E). A cap
becomes apparent at stage 10 when the surface is still not closed (F). The appearance of papillae covering the stigmatic surface defines
the start of stage 11 (G). Bars = 10 /urn.
(H) to (J) Structure of developing nectaries (lateral sepal removed). These first appear at the base of the lateral stamens in stage 9 (H)
and grow outward during stages 10 and 11 (I). They reach maturity when the flower opens and reaches stage 14 (J). Stomata often
occur at their apex. The righthand nectary in (J) has shrunken, probably because it has already released its nectar. Bars =10 ^m.
766 The Plant Cell

1989; Hill and Lord, 1989). The description of the surface

Table 3. Cumulative Totals of Flowers per Plant on 20 Plants
characteristics of normal organs (the present study; Bow-
Scored Each Day from 25 to 31 Days after lncubation at 25OC
man et al., 1989; Hill and Lord, 1989; Kunst et al., 1989)
also allows the effects of mutational changes, including Mean No. of Flowers
Mean No. of
the nature of mosaic structures, to be inferred. per Plant at Stages
Flowers Advancing
Shown
When these and other genes are cloned, the site and
time of their expression can be determined directly. In this Age lnto Out of
way we may ultimately understand how gene activity (davs)
~
6-12
~
13-17 Total
~
Stacle 6 Staae 12
directs flower development. 25 15.9 0.5 16.4 - -
26 16.0 2.2 18.1 1.7 1.7
27 16.7 3.8 20.5 2.4 1.6
METHODS
28 16.9 5.3 22.2 1.7 1.5
29 16.7 7.4 24.1 1.9 2.1
All plants were of the Landsberg ecotype and were homozygous 30 16.2 8.9 25.1 1.0 1.5
for the erecta mutation, which reduces plant height. Seeds were 31 15.6 10.6 26.2 1.1 1.7
planted on the surface of a mixture of 3 volumes of commercial
potting mix, 3 volumes of peat, and one of sand, spaced at about
4 cm2 per plant. They were treated at 4°C for 2 days and then
transferredto a growth chamber. The age of a plant was recorded
advice. This study was assisted by National Science Foundation
from the time of this transfer. Conditions were held at 25°C and
grant PCM-8703439 to E.M.M. J.L.B. is supported by training
70% relative humidity, and plants were grown under continuous
grant 5T32-GM07616 of the National lnstitutes of Health.
light provided by cool-white fluorescent tubes.
For scanning electron microscopy, whole inflorescences were
fixed in 3% glutaraldehyde in 0.025 M sodium phosphate buffer
(pH 6.8) at 4°C for at least 12 hr. They were then rinsed in the
buffer and further fixed in 1% OsO, in 0.05 M sodium cacodylate Received May 16, 1990
buffer (pH 7.0) for severa1 hours before dehydration through a
graded ethanol series. lnflorescences were then critical point dried
using CO,. Whole inflorescences were mounted on stubs and
shadowed with gold and palladium (4:l) before viewing with an REFERENCES
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 Early Flower Development in Arabidopsis 767

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