Download Ectopic Expression of AINTEGUMENTA in Arabidopsis Plants

DEVELOPMENTAL GENETICS 25:224–236 (1999)
Ectopic Expression of AINTEGUMENTA in
Arabidopsis Plants Results in Increased Growth of
Floral Organs
BETH ALLYN KRIZEK*
Department of Biological Sciences, University of South Carolina, Columbia, South Carolina
ABSTRACT
AINTEGUMENTA (ANT) was previously shown to be involved in floral organ initiation and
growth in Arabidopsis. ant flowers have fewer and
smaller floral organs and possess ovules that lack integuments and a functional embryo sac. The present work
shows that young floral meristems of ant plants are
smaller than those in wild type. Failure to initiate the full
number of organ primordia in ant flowers may result from
insufficient numbers of meristematic cells. The decreased
size of ant floral organs appears to be a consequence of
decreased cell division within organ primordia. Ectopic
expression of ANT under the control of the constitutive
35S promoter results in the development of larger floral
organs. The number and shape of these organs is not
altered and the size of vegetative organs is normal.
Microscopic and molecular analyses indicate that the
increased size of 35S::ANT sepals is the result of
increased cell division, whereas the increased sizes of
35S::ANT petals, stamens, and carpels are primarily
attributable to increased cell expansion. In addition,
35S::ANT ovules often exhibit increased growth of the
nucellus and the funiculus. These results suggest that ANT
stimulates cell growth in floral organs. Dev. Genet.
25:224–236, 1999. r 1999 Wiley-Liss, Inc.
Key words: Arabidopsis flower development; AINTEGUMENTA; cell division; cell expansion; AP2/EREBP
family
INTRODUCTION
Flowers are derived from groups of undifferentiated
cells called floral meristems. Floral organ primordia
arise at defined positions from within these meristems,
grow, and eventually differentiate into the four organs
of a flower (sepals, petals, stamens, and carpels). As
plant cells do not undergo migration, cell division and
cell expansion are the predominant mechanisms by
which the number and position of organ primordia is
determined. In addition, the final size and shape of each
organ is also controlled by these processes. The number,
location, and plane of each cell division in the developing organ primordia, as well as the amount and direction of cell expansion, are critically important in deter-
r 1999 WILEY-LISS, INC.
mining the final form of each organ. Although certain
aspects of flower development, such as the establishment of floral organ identity, are well characterized
[reviewed in Coen and Meyerowitz, 1991; Ma, 1994;
Sessions et al., 1998; Weigel and Meyerowitz, 1994],
very little is known about how the patterns and numbers of cell divisions are controlled in flowers during
organ initiation and organ growth [Meyerowitz, 1997].
Furthermore, little is known about the control of cell
expansion during plant development. Cell expansion is
known to be regulated by phytohormones such as
auxin, gibberellin, and brassinosteroids [reviewed in
Cleland, 1987; Hooley, 1996; Métraux, 1987]; mutants
in the biosynthesis or signal transduction pathways of
these hormones often exhibit defects in cell expansion.
Recently, additional genes with roles in controlling cell
expansion have been identified [Hanzawa et al., 1997;
Kim et al., 1998; Sablowski and Meyerowitz, 1998;
Wilson et al., 1996]. Whereas some genes seem to play
general roles in cell expansion in all tissues [Takahashi
et al., 1995], others appear to have specific functions in
particular organs [Sablowski and Meyerowitz, 1998] or
at particular times in development [Hanzawa et al.,
1997], suggesting that cell expansion is controlled by
different factors in different tissues.
One gene that is involved in the control of organ
growth during Arabidopsis flower development is AINTEGUMENTA (ANT). Mutations in ant result in a
random reduction in floral organ number, the production of narrow floral organs, and defects in ovule
development including the absence of integuments and
a female gametophyte [Baker et al., 1997; Elliott et al.,
1996; Klucher et al., 1996; Schneitz et al., 1997]. These
defects in both organ initiation and organ growth
suggest that ANT may be involved in regulating cell
Contract grant sponsor: Department of Energy; Contract grant number: 98ER20312.
*Correspondence to: Beth Allyn Krizek, Department of Biological
Sciences, University of South Carolina, Columbia, SC 29208.
E-mail: [email protected]
Received 3 March 1999; Accepted 1 June 1999
ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH
division in flowers. ANT is a member of the AP2/EREBP
family of transcription factors, containing two AP2
domains of approximately 70 amino acids [Elliott et al.,
1996; Klucher et al., 1996]. These domains have been
shown to bind DNA in other members of the AP2/
EREBP family [Buttner and Singh, 1997; Kagaya et al.,
1999; Liu et al., 1998; Ohme-Takagi and Shinshi, 1995;
Stockinger et al., 1997; Zhou et al., 1997]. In addition,
ANT has been shown to function as a transcription
factor in yeast [Vergani et al., 1997].
ANT is expressed in cotyledon, leaf, floral organ, and
ovule primordia [Elliott et al., 1996; Klucher et al.,
1996]. In flowers, ANT RNA is initially detected throughout organ primordia but later becomes restricted to
particular subdomains within developing organs [Elliott et al., 1996]. In particular, these domains of ANT
expression seem to correlate with regions undergoing
active growth [Elliott et al., 1996]. Ovule primordia
consist of several morphological regions: the base or
stalk of the primordia (funiculus) that connects the
ovule to the maternal tissue, a central or chalazal
region from which two integuments arise and eventually develop into the seed coat, and the apical tip of the
primordia (the nucellus) in which the megaspore mother
cell is produced [reviewed in Reiser and Fischer, 1993].
In ovules, ANT is initially expressed throughout the
primordia and later becomes restricted primarily to the
chalazal region of the ovule before integument initiation [Elliott et al., 1996]. Expression continues in the
integuments during their early development, decreases
as the outer integument grows to cover the nucellus,
and eventually becomes limited to the interiormost cell
layer of the inner integument [Elliott et al., 1996].
To investigate further the role of ANT in floral organ
initiation and growth, ANT was ectopically expressed
in wild-type Arabidopsis plants under the constitutive
cauliflower mosaic virus 35S promoter (35S::ANT). The
most dramatic phenotype exhibited by 35S::ANT plants
is the production of larger floral organs. The increased
size of these organs results from an increase in cell
number in the case of sepals and appears to be largely
due to an increase in cell size in petals, stamens, and
carpels. The relative shape of these organs is maintained, suggesting that organ size is controlled independently from organ shape. In addition, 35S::ANT ovules
often exhibit increased growth of the nucellus and
funiculus but decreased growth of the outer integument. Characterization of ant flowers by two photon
fluorescence microscopy shows that ant stage 3 floral
meristems are smaller than wild-type meristems of
similar age. Such data provide a possible explanation
for the decreased numbers of organs initiated in ant
mutants. The smaller size of ant floral organs results
from decreased cell division within floral organ primordia. Both the ant mutant and 35S::ANT phenotypes can
be explained by a model in which ANT stimulates cell
growth.
225
MATERIALS AND METHODS
Production of 35S::ANT Plants
ANT cDNA was PCR amplified using Pfu polymerase
(Stratagene) with primers containing BamHI and XbaI
restriction sites at the 58 and 38 ends of the cDNA,
respectively. The polymerase chain reaction (PCR) product was originally cloned into pLITMUS28 (New England Biolabs), and its sequence was verified by doublestranded sequencing of the recombinant plasmid. ANT
was subsequently subcloned into pGEM3Z containing
the 35S promoter in the KpnI/BamHI sites. 35S::ANT
was cut out of this plasmid with KpnI/XbaI and subcloned into the plant transformation vector pCGN1547,
which contained a 38 NOS sequence [Krizek and Meyerowitz, 1996]. 35S::ANT/pCGN-NOS was transformed
into Agrobacterium ASE by electroporation and subsequently transformed into L-er, ant ⫺6/⫹, and ant ⫺8/⫹
plants using the in planta vacuum infiltration procedure [Bechtold et al., 1993]. Transformants were selected by germination of the seeds on MS media containing kanamycin. Putative 35S::ANT ant ⫺6 and 35S::
ANT ant ⫺8 plants were genotyped as described later
in Materials and Methods.
Organ Length, Cell Length, and Cell Area
Measurements
Floral organ lengths were measured using an ocular
micrometer. Two adjacent sepals, two adjacent petals,
and two lateral stamens were removed from stage 14
flowers (flowers staged as described in Smyth et al.
[1990] and Müller [1961]). The length of the carpel was
measured after the remaining floral organs were removed from each flower. The average size of petal blade
cells from L-er and 35S::ANT flowers was determined
using the software IPLab (Scanalytics, Fairfax, VA).
Scanning electron micrograph images were segmented
into individual cells for quantitation by either hand
drawing around the cell or using intensity thresholding. The areas of segments corresponding to individual
petal cells were then calculated by IPLab. The average
length of anther epidermal cells also was determined
using IPLab. Lines were drawn the middle of each of 13
cells from scanning electron micrographs of L-er and
35S::ANT anthers. The lengths of these lines were then
calculated by IPLab. For the petal and stamen IPLab
measurements, four to five SEM image files from four to
five different petals or stamens, respectively, were used.
The images used in these measurements corresponded
to similar parts of the respective floral organs in the
L-er and 35S::ANT flowers.
Scanning Electron Microscopy and Two Photon
Fluorescence Microscopy
Samples for SEM were fixed and dried as described
previously [Bowman et al., 1991]. For viewing ovules,
carpels were sliced with a razor blade immediately
before fixation. Flowers and floral organs were mounted
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KRIZEK
onto stubs, dissected with glass needles as necessary,
and coated with gold in a Denton Desk II gold sputter/
etch unit. Images were collected on a Hitachi S-2500D
scanning electron microscope and digitally saved using
the software Iridium (IXRF Systems, Houston, TX).
Inflorescence tissue from L-er, 35S::ANT, and ant ⫺5
plants was prepared for two photon microscopy as
described previously for confocal microscopy [Running
et al., 1995], except that in some cases, the tissue was
treated with RNase at 50 mg/ml for 30 min at 37°C after
fixation and before staining in propidium iodide. This
step reduced staining in the cytoplasm. The data were
collected on a Bio-Rad system equipped with a titaniumsapphire laser using a ⫻60 oil immersion lens. Propidium iodide was excited at 770 nm, and emitted light
was collected after passage through filters absorbing
wavelengths shorter than 575 nm.
Sequencing and Genotyping of the ant ⴚ6 and
ant ⴚ8 Alleles
DNA from ant ⫺6 and ant ⫺8 plants [Baker et al.,
1997] was isolated using standard methods. ant was
PCR amplified from each of these DNA samples in five
overlapping pieces using either Pfu (Stratagene) or
vent (New England Biolabs) polymerase. These PCR
products were then sequenced directly on a ABI 377
automated sequencer. The ant ⫺6 mutation C679=T
results in a nonsense mutation (Gln227=stop codon).
An exon 2 primer was engineered to create an MseI site
specifically in the ant ⫺6 allele. PCR was performed on
leaf tissue [Klimyuk et al., 1993] removed from putative
35S::ANT ant ⫺6 plants using this primer and an
intron 2 primer. The ant ⫺8 mutation (G1267=A,
which results in an Ala =Thr missense mutation)
disrupts a PstI site. An intron 7-specific primer and
exon 8 primer were used to PCR-amplify this region
from candidate 35S::ANT ant ⫺8 plants. Restriction
enzyme analyses distinguished plants that were homozygous wild-type, homozygous ant ⫺6 (or ant ⫺8), or
heterozygous.
In Situ Hybridization
Flowers for radioactive ANT in situs and nonradioactive histone H4 in situs were fixed, embedded, sectioned, and hybridized as described previously [Sakai et
al., 1995]. The radioactive ANT in situ slides were
washed as described previously [Sakai et al., 1995]. To
make the ANT antisense probe, a BamHI/ClaI fragment of ANT cDNA (corresponding to the 58 half of the
gene, not including the AP2 repeats) was cloned into the
HincII site of pGEM3Z (pANTsitu). pANTsitu was
linearized by digestion with EcoRI and in vitro transcribed in the presence of [35S]UTP with SP6 RNA
polymerase. Sections were exposed for 1–2 weeks.
Flowers for the nonradioactive histone H4 in situs were
washed as previously described [Coen et al., 1990].
Immunological detection of the hybridized probe was
performed by blocking with 1% Boehringer blocking
agent in phosphate-buffered saline (PBS) with 0.3%
Triton X1000 for 1 h, blocking with 0.5% bovine serum
albumin (BSA) in PBS with 0.3% Triton X1000 for 1 h,
incubation in anti-DIG antibody diluted 1:1,000 in 0.5%
BSA/PBS/0.3% Triton X1000 for 5 h, washing with PBS
and subsequently 100 mM Tris pH9.5, 10 mM NaCl, 50
mM MgCl2, and color development with NBT/X-phos
for approximately 12 h. Histone H4 antisense probe
was made by linearization of pHS-H4 with SpeI and in
vitro transcription with T7 RNA polymerase in the
presence of digoxigenin-11-UTP (Boehringer-Mannheim). Histone H4 sense probe was made by linearization of pHS-H4 with NotI and in vitro transcription
with SP6 RNA polymerase in the presence of digoxigenin-11-UTP. The number of cells expressing histone
H4 in sepals and petals (of stage 7 and 10 flowers) was
determined in the following manner. Adjacent tissue
sections of appropriately staged flowers that contained
a full-length longitudinal section of a sepal or petal
were identified, the number of stained cells in each
tissue section counted, and an average for that individual organ determined. An overall average was calculated from these individual organ averages.
RESULTS
Ectopic Expression of ANT Increases Floral
Organ Size
After initial expression throughout floral organ primordia, ANT RNA becomes limited to particular subdomains as the organs mature [Elliott et al., 1996] (Fig.
1A–D). To investigate the consequences of changing the
levels and/or duration of ANT expression in developing
organ primordia, ANT cDNA was fused to the constitutive 35S promoter from cauliflower mosaic virus and
this construct was transformed into wild-type Arabidopsis plants. As shown in Figure 1E–H, 35S::ANT confers
persistent ANT expression in developing floral organs.
Approximately one-half of the transgenic plants (66 out
of 127 plants) produced larger than normal flowers (Fig.
2A). Of the other transformants, most were wild-type in
appearance (58), while three had phenotypes resembling ant mutants. The plants with ant phenotypes may
arise from cosuppression [reviewed in Depicker and
van Montagu, 1997]. Further characterization was
performed on those plants which produced the larger
flower phenotype. Because these plants are sterile
(described later), all phenotypic characterizations were
done on primary transformants. DNA gel blot analysis
indicated that some of the 35S::ANT lines with the
larger flower phenotype contained a single copy of the
transgene, whereas others had multiple copies.
The increased size of these 35S::ANT flowers is
attributable to increases in the size of all four types of
floral organs. Floral organ number is unchanged in
35S::ANT plants. The differences in organ size were
quantitated by measuring the lengths of floral organs
removed from L-er and 35S::ANT stage 14 flowers
ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH
227
Fig. 1. Expression pattern of ANT in L-er and 35S::ANT plants. In
situ hybridization of an ANT antisense RNA probe with longitudinal
sections through wild-type (L-er) (A–D) and 35S::ANT tissue (E–H).
Each section was photographed in brightfield (A,C,E,G) or darkfield
(B,D,F,H). A,B: ANT RNA is detected throughout young floral primor-
dia (arrow). Expression decreases in older flowers. C,D: ANT is no
longer expressed in the sepals or anthers of this stage 10 flower. E,F:
ANT RNA is detected throughout the inflorescence of 35S::ANT
plants. G,H: ANT RNA is detected throughout this stage 9 35S::ANT
flower. se, sepal; pe, petal; st, stamen; ca, carpel.
(flowers staged as described in Smyth et al. [1990] and
Müller [1961]), using a optical micrometer. These data
were collected from the first 10 flowers on L-er and
35S::ANT plants that were grown side by side under
equivalent conditions. Results from two different experiments are shown in Table 1. Organ size is increased
from 11–34% in 35S::ANT flowers. The increased size of
35S::ANT floral organs is the opposite phenotype of
that produced by mutations in ant which cause the
development of smaller floral organs [Elliott et al.,
1996; Klucher et al., 1996]. Although organ width was
not measured, the overall shape of the larger 35S::ANT
floral organs is normal (Fig. 2B). This is not true of
mutations in ant, which tend to produce quite narrow
floral organs [Baker et al., 1997; Elliott et al., 1996;
Klucher et al., 1996] (Fig. 2B).
The effects of ectopic ANT expression on organ growth
appear to be restricted to floral organs as no differences
were observed in the size of leaves on 35S::ANT plants
compared with L-er plants or in the height of 35S::ANT
plants compared with L-er plants (Fig. 2C). To confirm
that ANT is expressed in these vegetative organs, in
situ hybridization was performed on 35S::ANT leaf
tissue sections. ANT RNA was detected throughout
35S::ANT leaf tissue (data not shown).
layed in flowering compared with 35S::ANT plants with
a wild-type appearance. Epicuticular wax that is normally present on ovary epidermal cells is not present on
the surface of these cells in 35S::ANT flowers. 35S::
ANT plants are male sterile and show severe reductions in female fertility. The anthers do not dehisce;
however, this is not the only cause of the male sterility
phenotype. A defect in a late stage of pollen development appears to occur in 35S::ANT lines. Microspores
with an exine wall are made and, in at least some cases,
viable pollen grains are produced. Pollen viability was
measured by staining with the dye fluorescein diacetate
(FDA) [Regan and Moffatt, 1990], which assays for the
integrity of the plasma membrane of the vegetative cell
[Heslop-Harrison and Heslop-Harrison, 1970]. However, manual cutting of mature 35S::ANT anthers does
not release individual dehydrated pollen grains as
observed for wild-type anthers. Further work will be
necessary to fully characterize pollen development in
35S::ANT flowers.
When wild-type pollen is used to fertilize 35S::ANT
carpels, a few seeds are occasionally produced. To
investigate the basis for this reduced female fertility,
the development of 35S::ANT ovules were characterized by scanning electron microscopy (SEM). The following discussion presents a brief description of wild-type
ovule development [Robinson-Beers et al., 1992;
Schneitz et al., 1995] using the stages assigned in
Schneitz et al., 1995. Finger-like ovule primordia are
initiated from the inner ovary walls during stage I of
ovule development. During stage II, the inner integu-
Other Floral Phenotypes Resulting From
Ectopic Expression of ANT
Several other effects on flower development were
observed in 35S::ANT plants that exhibit the larger
floral organ phenotype. These plants are slightly de-
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KRIZEK
Fig. 2. Phenotypes of 35S::ANT, 35S::ANT ant ⫺6, and 35S::ANT
ant ⫺8 plants. A: Top down and side views of wild-type (L-er) and
35S::ANT flowers. B: Petals removed from L-er, 35S::ANT, and ant ⫺9
flowers. ant ⫺9 is a strong allele. C: L-er and 35S::ANT plants of equal
age. The size of vegetative organs and the overall stature of these
plants are the same. D: ant ⫺6 and 35S::ANT ant ⫺6 flowers showing
the lack of complementation of the organ number and size defects of
ant ⫺6. E: Scanning electron micrograph of a typical ant ⫺6 ovule.
There is a slight expansion in the chalazal region of these ovules,
where the inner and outer integuments initiate in wild-type ovules. F:
Scanning electron micrograph of a 35S::ANT ant ⫺6 ovule showing
increased growth of a single integument-like structure. G: Scanning
electron micrograph of an ant ⫺8 ovule showing partial growth of a
single integumentary-like structure around the nucellus. H: ant ⫺8
and 35S::ANT ant ⫺8 flowers showing complementation of the ant ⫺8
mutation by 35S::ANT. Scale bars ⫽ 10 µm (E–G).
ment initiates as a symmetrical ring of cells in the
middle or chalazal region of the ovule primordia. The
outer integument initiates soon afterward as an asymmetrical extension of cells on the abaxial surface of the
ovule just below the inner integument (Fig. 3A,B).
During stages II and III, the inner integument expands
symmetrically to cover the nucellus while the outer
integument grows asymmetrically with more growth on
the abaxial surface than the adaxial surface of the
ovule. The outer integument eventually grows to cover
the inner integument (Fig. 3C) and finally the nucellus
(Fig. 3D).
Differences in the development of 35S::ANT ovules
are apparent shortly after the initiation of ovule primordia. Although two integuments are initiated in 35S::
ANT ovules, they are not as well defined as those in
wild-type ovules (Fig. 3E,F). This is particularly true
for the inner integument, which does not protrude
significantly from the chalazal region of the ovule
primordia (Fig. 3E). In 35S::ANT ovules, the outer
integument appears to overtake the inner integument
at a slightly earlier time than in wild-type ovules (cf.
Fig. 3C,G). Unlike wild-type ovules, in which the outer
integument completely surrounds the nucellus during
ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH
TABLE 1. Floral Organ Lengths (mm) in L-er and
35S⬋ANT Flowers*
Sepals
Petals
Stamens
Carpels
L-er
1.86 ⫾ 0.08 3.15 ⫾ 0.21 2.51 ⫾ 0.16 2.49 ⫾ 0.21
35S⬋ANT 2.28 ⫾ 0.13 3.95 ⫾ 0.27 2.79 ⫾ 0.25 3.17 ⫾ 0.22
% increase
22.7%
25.2%
11.1%
27.6%
L-er
1.88 ⫾ 0.14 3.11 ⫾ 0.38 2.65 ⫾ 0.21 2.54 ⫾ 0.31
35S⬋ANT 2.37 ⫾ 0.11 4.16 ⫾ 0.25 3.02 ⫾ 0.29 3.36 ⫾ 0.19
% increase
25.9%
34.0%
14.1%
32.0%
*The data are indicated as averages ⫾ SD. The first data set is
based on measurements on two sepals, two petals, two stamens, and one carpel from each of 28 L-er flowers on four
plants and each of 45 35S⬋ANT flowers on 14 plants located
at positions 1–10 on the inflorescence. The second data set is
based on measurements on two sepals, two petals, two stamens, and one carpel from each of 29 L-er flowers on 4 plants
and each of 47 35S⬋ANT flowers on eight plants located at
positions 1–10 on the inflorescence.
stage III, the outer integuments of 35S::ANT ovules
rarely grow to surround the nucellus entirely (cf. Fig.
3D,H).
A small number (approximately 10%) of mature
35S::ANT ovules are wild-type in appearance (cf. Fig. 3I
and J), whereas the rest exhibit growth defects in the
different ovule structures. In most 35S::ANT ovules,
growth of the outer integument is prematurely terminated such that the nucellus is still visible (Fig. 3K).
Some of these 35S::ANT ovules resemble ovules of the
weak ant ⫺3 allele [Klucher et al., 1996]; however, in
many cases, an unusually large nucellus protrudes
from the reduced outer integument (Fig. 3L–N). Occasionally, ovules with a similar appearance were identified that had a micropyle-like hole visible in the tissue
protruding from the outer integument (Fig. 3Q). This
finding suggests that the protruding tissue is the inner
integument; although analysis of the development of
35S::ANT ovules indicated that the inner integument
stops growing before the outer integument. As this class
of ovule was only observed rarely, it is possible that
such a phenotype was missed during examination of
early ovule development because of the smaller number
of ovules examined. A few 35S::ANT ovules resemble
sup ovules [Gaiser et al., 1995] in which there is
increased growth of the outer integument on the adaxial surface of the ovule (Fig. 3H). In addition to the
defects noted above, the funiculus of 35S::ANT ovules is
often increased in length (Fig. 3N–P). This increased
growth appears to primarily result from increased cell
elongation (cf. Fig. 3I and P).
35S::ANT Sepals Contain More Cells, Whereas
35S::ANT Petals, Stamens, and Carpels Contain
Larger Cells
The increased size of 35S::ANT floral organs could
result from an increase in cell number, an increase in
cell size, or some combination of the two. SEM was used
to characterize the size of sepal, petal, stamen, and
229
carpel epidermal cells in wild-type and 35S::ANT flowers. Comparison of cell size in sepals, petals, and
carpels was performed on stage 14 flowers, at which
time dehiscence of the anthers had already occurred.
Stamen cell size was compared in stage 13 flowers in
which the anthers had just begun to dehisce. Epidermal
cells of petals, stamens, and carpels were found to be
larger in 35S::ANT flowers compared with L-er flowers
(cf. Fig. 4B–E and G–J). Thus, the increased size of
petals, stamen, and carpels in 35S::ANT flowers is at
least partially attributable to the presence of larger
cells. No obvious difference in cell size was apparent in
sepals (Fig. 4A,F), indicating that ectopic expression of
ANT results in an increased number of cells in sepals.
In general, cell shape and epidermal characteristics are
conserved.
To determine whether the increased size of cells in
petals and stamens could account entirely for the
increased size of these organs, scanning electron micrographs were analyzed with the graphics software IPLab.
The cell area of both the adaxial and abaxial petal blade
cells and the length of anther epidermal cells were
determined (Table 2). The increase in the average cell
area of adaxial and abaxial petal blade cells in 35S::
ANT flowers is approximately equal to the increase in
petal size of 35S::ANT flowers, suggesting that increased cell expansion can account entirely for the
increased size of these organs. An increase in the
average length of 35S::ANT anther epidermal cells was
similar to the overall increase in stamen length, suggesting that increased cell expansion may also account for
the increased length of stamens in 35S::ANT flowers,
although the relative size of cells in the stamen filament was not determined.
Histone H4 Expression in L-er and
35S::ANT Flowers
To investigate further whether the larger size of
35S::ANT sepals is due to increased cell division, the
expression pattern of a cell division specific marker was
examined in L-er and 35S::ANT sepals. Histone H4 has
previously been shown to be expressed specifically
during interphase in Antirrhinum floral meristem cells
[Fobert et al., 1994] and predominantly during S phase
of the cell cycle in many organisms [Marzluff and
Pandey, 1988; Nakayama and Iwabuchi, 1993]. Overall,
the pattern of histone H4 expression is similar in L-er
and 35S::ANT flowers. In both L-er and 35S::ANT
inflorescences and flowers, histone H4 is expressed in a
spotty pattern, with each spot corresponding to an
individual cell or a small group of cells (Fig. 5A–D). In
sepals, petals, and stamens of older flowers (Fig. 5B,D),
fewer cells are labeled, indicating decreased numbers of
dividing cells or decreased rates of cell division, or both.
These observations are similar to what has been reported previously for Antirrhinum [Fobert et al., 1994].
No signal was detected with a histone H4 sense probe.
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KRIZEK
Fig. 3. Scanning electron micrographs of ovule development in L-er
and 35S::ANT flowers. Wild-type ovule development (A–D) and a
mature wild-type ovule (I). 35S::ANT ovule development (E–H) and
mature 35S::ANT ovules (J-P). Ovules have been staged according to
the descriptions of Schneitz et al., 1995. A: Wild-type ovules at stage
2-IV. The inner integument and outer integument have initiated. B:
Wild-type ovules at stage 2-V. Both integuments are growing around
the nucellus. C: Stage 3-I wild-type ovules in which the outer
integument is overtaking the inner integument. D: Stage 3-I wild-type
ovules in which the outer integuments have enclosed the nucellus. E:
35S::ANT ovules in an early stage of development. Both the inner and
outer integuments have initiated although they are not as well defined
as in wild-type ovules. F: 35S::ANT ovules at stage 2-V, in which the
outer integument is beginning to cover the inner integument. G: Stage
3-I 35S::ANT ovules in which the outer integument has completely
enclosed the inner integument. H: Stage 3-I 35S::ANT ovules. In some
cases, the nucellus continues to protrude from the outer integument. I:
Mature wild-type ovule. J: 35S::ANT mature ovule that has a fairly
normal morphology. K: 35S::ANT ovule in which the outer integument
has not grown to fully enclose the nucellus. L: 35S::ANT ovule in
which the outer integument does not fully enclose the nucellus and the
nucellus has grown abnormally large. M: 35S::ANT ovules with a large
nucellus protruding from the outer integument. N: 35S::ANT ovule
with an enlarged nucellus and a long funiculus. O: 35S::ANT ovule in
which the protruding tissue has an opening resembling a micropyle,
suggesting that this is the inner integument. P: 35S::ANT ovule in
which the outer integument shows increased growth on the adaxial
side of the ovule, resembling sup ovules. ii, inner integument; oi, outer
integument; n, nucellus; f, funiculus; m, micropyle. Scale bars ⫽
10 µm.
Fig. 4. Scanning electron micrographs of the epidermal cells on L-er and 35S::ANT floral
organs. Epidermal cells from L-er organs are shown in A–E and corresponding epidermal
cells (at the same magnification) from 35S::ANT organs are shown in F–J. A: L-er sepal
epidermal cells. B: Epidermal cells on the adaxial surface of a L-er petal. C: Epidermal cells
on the abaxial surface of a L-er petal. D: Anther epidermal cells from a L-er stamen. E:
Ovary epidermal cells from a L-er carpel. F: 35S::ANT sepal epidermal cells. G: Epidermal
cells on the adaxial surface of a 35S::ANT petal. H: Epidermal cells on the abaxial surface of
a 35S::ANT petal. I: Anther epidermal cells from a 35S::ANT stamen. J: Ovary epidermal
cells from a 35S::ANT carpel. Scale bars ⫽ 10 µm.
232
KRIZEK
TABLE 2. Average Cell Size (Petals) and Average Cell
Length (Stamens) in L-er and 35S⬋ANT Floral Organs*
L-er
35S⬋ANT
% increase
Petals
(adaxial)
1940 ⫾ 727
2458 ⫾ 746
26.7%
Petals
(abaxial)
1690 ⫾ 471
2245 ⫾ 496
32.8%
Stamens
102 ⫾ 21
113 ⫾ 20
10.8%
*The data are indicated as averages ⫾ SD. Measurement
units are pixels. The adaxial petal data are based on measurements using 189 L-er and 259 35S⬋ANT cells from a total of
four (L-er) or five petals (35S⬋ANT). The abaxial petal data
are based on measurements using 132 L-er and 118 35S⬋ANT
cells from a total of four petals each. The stamen data are
based on measurements using 52 L-er and 52 35S⬋ANT cells
from four stamens of each.
The number of histone H4-expressing cells in sepals
of L-er and 35S::ANT flowers of stages 6, 7, and 10 was
counted (Table 3). Similar numbers of sepal cells expressing histone H4 were found in L-er and 35S::ANT
stage 6 and 7 flowers. However, in sepals from stage 10
flowers, more than twice as many 35S::ANT cells
expressed histone H4 as compared with L-er cells.
These results are consistent with the SEM data providing further evidence that increased cell division accounts for the increased size of 35S::ANT sepals. These
results do not indicate whether the rate of cell division
in stage 10 35S::ANT sepals is faster than in wild-type
sepals or whether the population of dividing cells is
larger. For comparison, the number of L-er and 35S::
ANT petal cells in stage 10 flowers that expressed
histone H4 was also determined. No significant differences were detected, consistent with earlier results
indicating that increased cell expansion is primarily
responsible for the increased size of 35S::ANT petals.
Inflorescence and Floral Meristem Size in L-er,
35S::ANT, and ant ⴚ5 Plants
To characterize further the effects of ectopic ANT
expression on floral organ growth, two photon fluorescence microscopy was used to compare the size of young
floral meristems and young floral organ primordia in
L-er and 35S::ANT plants. Stage 3 floral meristems of
L-er and 35S::ANT plants are similar in size (Fig.
6A,D), indicating that the increase in organ growth is
not a consequence of a larger floral meristem. In
addition, the sepal primordia are approximately the
same size in both L-er and 35S::ANT flowers suggesting
that 35S::ANT sepals do not initially consist of more
cells. The height and width of stamen and carpel
primordia in stage 6 (Fig. 6B,E) and stage 7 (Fig. 6C,F)
L-er and 35S::ANT flowers is also quite similar. These
data provide additional evidence that the predominant
effects of ectopic ANT expression on stamens and
carpels is increased cell expansion during later stages
of floral organ development. The effects of ant mutations on floral meristem and organ growth were also
investigated by two photon fluorescence microscopy.
Stage 3 ant floral meristems are not as wide as wildtype stage 3 meristems (Fig. 6G). The average width of
ant ⫺5 floral meristems was 40 ⫾ 4 mm, while that of
wild type was 54 ⫾ 1 mm (average ⫾ standard deviation). The carpel primordia in a stage 6 ant ⫺5 flower
(Fig. 6H) are approximately the same size as those in a
L-er flower. By stage 7, both stamen and carpel primordia in ant ⫺5 flowers are thinner than those in wildtype flowers and appear to consist of fewer cell layers
(Fig. 6I). These ant ⫺5 organ primordia have grown
more in length than width, changing the shape of the
primordia and making them somewhat difficult to
stage. These results are consistent with mutations in
ant affecting both the numbers and patterns of cell
divisions in flowers. SEM analysis of ant ⫺5 mature
floral organs indicates that in almost all cases, the size
of epidermal cells are the same as in L-er flowers (data
not shown).
Ectopic Expression of ANT in ant ⴚ6 and ant ⴚ8
Because ectopic expression of ANT sometimes results
in ovule defects similar to those observed in weak ant
mutants, the ability of 35S::ANT to complement mutations in ant was investigated. Intermediate and weak
ant alleles (ant ⫺6 and ant ⫺8, respectively) were used
in these studies. ant ⫺6 contains the nucleotide substitution C679=T which converts Gln227 into a stop
codon, while ant ⫺8 contains the nucleotide substitution G1267=A, which converts Ala422 into Thr. Interestingly, the ant ⫺6 allele displays an intermediate
phenotype with slightly more expansion in the chalazal
region of the ovule primordia than found in strong ant
alleles (Fig. 2E) [Baker et al., 1997], yet contains a stop
codon before the predicted AP2 repeat DNA binding
domains. The Ala that is mutated in ant ⫺8 is conserved among different members of the AP2 protein
family [Okamuro et al., 1997; Riechmann and Meyerowitz, 1998]. Since 35S::ANT plants are male sterile and
mostly female sterile, the 35S::ANT construct was
transformed into ant ⫺6/⫹ and ant ⫺8/⫹ plants. Kanamycin resistant progeny from these transformations
were PCR genotyped [Jacobson and Moscovits, 1991].
Five 35S::ANT ant ⫺6 plants were obtained which all
resembled ant ⫺6 plants with regard to floral organ
number and floral organ size (Fig. 2D). However, a
single integumentary-like structure grew around the
nucellus in 35S::ANT ant ⫺6 ovules that is not present
in ant ⫺6 ovules (cf. Fig. 2E,F). These 35S::ANT ant ⫺6
ovules closely resemble ovules found in the weak ant
⫺8 allele (Fig. 2G) [Baker et al., 1997]. Five 35S::ANT
ant ⫺8 plants were obtained. Three of these plants
resembled 35S::ANT plants with regard to floral organ
number and size (Fig. 2H), while two resembled ant ⫺8
plants. The three lines with 35S::ANT-like larger flowers also mimicked the other 35S::ANT phenotypes
(anther and ovule defects and the absence of epicuticular wax). Thus, ectopic expression of ANT is able to
partially rescue the ovule defect of ant ⫺6 plants and
ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH
233
Fig. 5. Expression of histone H4 in L-er and 35S::ANT flowers. L-er (A,B) and 35S::ANT (C,D). Small
arrows, some of the histone H4 expressing cells in sepals. Wider arrows, petals of stage 10 flowers (B,D).
A: Stage 6 L-er flower. B: Stage 10 L-er flower. C: Stage 6 35S::ANT flower. D: Stage 10 35S::ANT flower.
se, sepal; pe, petal.
TABLE 3. Number of Cells Expressing Histone H4 RNA
in L-er and 35S⬋ANT Flowers*
L-er
35S⬋ANT
Stage 6
sepals
7.3 ⫾ 2.1
8.1 ⫾ 2.5
Stage 7
sepals
9.6 ⫾ 4.1
9.1 ⫾ 3.1
Stage 10
sepals
2.1 ⫾ 1.5
4.8 ⫾ 1.8
Stage 10
petals
21.1 ⫾ 6.9
22.6 ⫾ 3.6
*The data are indicated as averages ⫾ SD. The stage 6 sepal
data are based on measurements from 12 sepals on 6 L-er
flowers and 22 sepals on 11 35S⬋ANT flowers. The stage 7
sepal data are based on measurements from 12 sepals on 6
L-er flowers and 12 sepals on 6 35S⬋ANT flowers. The stage
10 sepal data are based on measurements from 17 sepals on 9
L-er flowers and 18 sepals on 9 35S⬋ANT flowers. The petal
data are based on measurements from 11 petals on 11 L-er
stage 10 flowers and 13 petals on 9 35S⬋ANT stage 10
flowers.
rescues both the floral organ and ovule defects of ant ⫺8
plants.
DISCUSSION
Ectopic Expression of ANT Results
in Larger Flowers
Ectopic expression of ANT under a constitutive promoter results in increased growth of floral organs. This
phenotype is the opposite of that resulting from loss of
ANT function; ant mutants produce small, narrow
floral organs [Baker et al., 1997; Elliott et al., 1996;
Klucher et al., 1996; Schneitz et al., 1997]. The increased growth of 35S::ANT floral organs is manifested
as increased cell division in sepals and increased cell
expansion in petals, stamens, and carpels. Owing to the
nature of the ANT expression pattern (broad initial
expression with subsequent restriction to particular
regions of developing organs), it is unclear whether the
increased growth of 35S::ANT floral organs results
from an increased level of ANT function and/or a
lengthened period of ANT activity in developing floral
organs. Some evidence for the latter of these possibilities is the lack of obvious size differences between L-er
and 35S::ANT floral buds of stage 7 and younger.
In ovules, ectopic expression of ANT results in increased growth of the proximal (funiculus) and distal
(nucellus) elements of an ovule primordia but in decreased growth of the integuments arising from the
chalazal region. In wild-type ovules, an initially broad
ANT expression domain throughout the primordium
becomes restricted to the central region of the primordium including the chalazal region and the distal part
of the funiculus [Elliott et al., 1996]. Thus, the increased growth of the nucellus and funiculus in 35S::
ANT ovules is likely to result from ectopic expression of
ANT in these regions of an ovule. The decreased growth
of the outer integument in these ovules may result from
some sort of integument-specific cosuppression of ANT.
Alternatively, growth of the outer integument may
requires a precise level of ANT expression. Differences
in the levels and patterns of ANT expression in 35S::
ANT floral meristems and organs may also explain the
inability of 35S::ANT to complement the ant ⫺6 mutation.
Fig. 6. Two photon fluorescence microscopy of flower development in L-er, 35S::ANT, and
ant ⫺5 flowers. L-er (A–C), 35S::ANT (D–F), and ant ⫺5 (G–I). A: L-er stage 3 flower, in
which the sepal primordia have initiated. B: L-er stage 6 flower. C: L-er stage 7 flower. D:
35S::ANT stage 3 flower. E: 35S::ANT stage 6 flower. F: 35S::ANT stage 7 flower. G: ant ⫺5
stage 3 flower. The floral meristem is not as wide as a similar stage wild-type floral
meristem. H: ant ⫺5 flower of approximately stage 6. While the carpel primordia are
similar in size to those of wild type, the stamen primordia appear slightly thinner than
stamen primordia of wild-type flowers. I: ant ⫺5 flower of approximately stage 7. Both the
stamen and carpel primordia are thinner those of wild-type; growing more in length than
width. Scale bars ⫽ 50 µm.
ECTOPIC ANT EXPRESSION INCREASES FLORAL ORGAN GROWTH
Comparison of ANT With Other Genes Involved
in Regulating Cell Expansion
The specificity of ANT ectopic expression on altering
cell growth in flowers is similar to ectopic expression of
another gene, NAP (NAC-like, activated by AP3/PI)
[Sablowski and Meyerowitz, 1998]. This gene was
identified as a target gene of the floral homeotic proteins APETALA3 (AP3) and PISTILLATA (PI) and is
expressed in regions of petals and stamens during later
floral stages (stage 8 to maturity). No NAP mutant has
been isolated, but expression of NAP under the 35S
promoter inhibits cell expansion in petals and stamens
resulting in shorter organs. Thus NAP and ANT have
opposite effects on cell growth in petals and stamens.
NAP has been postulated to function in petals and
stamens during the transition from organ growth resulting from cell division to that resulting from cell expansion [Sablowski and Meyerowitz, 1998].
Besides ANT, another member of the AP2/EREBP
family of transcription factors has been shown to be
involved in cell growth [Wilson et al., 1996]. The TINY
gene seems to play a general role in promoting cell
expansion. tiny plants are short in height, with stem
epidermal cells that are reduced in length. tiny flowers
have shorter stamens and pistils and rounder buds.
Leaf epidermal cells from tiny have a different shape
and are thicker than wild-type cells. Because tiny was
identified as a dominant mutant in a screen using a Ds
transposon containing a 35S promoter, the tiny phenotype is thought to arise from ectopic expression or
overexpression of the gene [Wilson et al., 1996]. Thus,
TINY and ANT appear to have opposite roles in cell
growth and differ with regard to the organs which are
affected and whether cell shape is altered. tiny, 35S::
NAP, and 35S::ANT plants all show reduced fertility
[Sablowski and Meyerowitz, 1998; Wilson et al., 1996].
Few mutants or transgenes have been identified that
increase organ growth. One mutant that causes increased growth of leaves and sometimes floral organs is
revoluta (rev) [Talbert et al., 1995]. In the case of leaves,
the increase in organ size is due to extra cell divisions,
but the basis for the larger floral organs was not
reported [Talbert et al., 1995]. Thus, it is unknown
whether rev affects cell expansion in addition to cell
division. The 35S::ANT transgene seems to be currently
unique in its ability to confer increased organ growth
that is specific to flowers. In addition, these plants
exhibit few other phenotypes, none of which occurs
during vegetative development. The ability to increase
carpel and ovary size through the use of the 35S::ANT
transgene might prove useful for producing larger
yields in agriculture.
Model for ANT Function
ANT is required for floral organ initiation and growth
[Baker et al., 1997; Elliott et al., 1996; Klucher et al.,
1996; Schneitz et al., 1997]. The results presented in
235
this paper indicate that ANT is sufficient in flowers for
organ growth but not organ initiation. The decreased
number of floral organs in ant mutants result from a
failure to initiate a normal number of organ primordia
and not from abortion of organ primordia after initiation. Since ant floral meristems are smaller than wild
type, the decrease in organ number may result from an
insufficient number of meristematic cells for incorporation into organ primordia. The decreased size of ant
floral organs results from changes in the number and
orientations of cell divisions within developing floral
organ primordia.
Considering both the loss of function ant mutant
phenotype as well as the 35S::ANT ectopic expression
phenotype, the following model describes how ANT
might control organ initiation and growth in flowers.
The model proposes that ANT promotes cell growth. In
the absence of ANT, cell growth is restricted. Studies in
yeast and other systems have shown that cells must
reach a critical size before dividing [Mitchison et al.,
1997]. Cells below this size threshold are prevented
from completing the cell cycle. Because of this close
relationship between cell growth and cell proliferation,
a decrease in cell growth can cause a reduction in cell
division. The decreased numbers of cells in ant mutants
is thus proposed to be a consequence of decreased cell
expansion. Decreased cell division in ant floral meristems and young floral organ primordia ultimately
leads to the presence of too few cells in young floral
meristems, a reduction in organ initiation, and the
development of smaller floral organs. In 35S::ANT
plants, increased ANT function stimulates cell growth.
This increased growth results in more cell divisions in
sepals. In petals, stamens, and carpels increased cell
growth is not accompanied by increased cell division. It
remians unclear why the effects of increased growth are
manifested in different ways in these different floral
organs. Perhaps other factors involved in carrying out
these growth processes are limiting in different organs.
ACKNOWLEDGMENTS
I thank Jannie Lee for doing the histone H4 in situs,
Hajime Sakai for pHS-H4, Charles Gasser for ant ⫺5,
ant ⫺6, ant ⫺7, and ant ⫺8 seeds, David Smyth for ant
⫺9 seeds and ANT cDNA, Dick Vogt for the use of his
compound microscope and photography system and for
help with two photon fluorescence microscopy, Madilyn
Fletcher’s laboratory for the use of IPLab, Dana Dunkelberger and Shawn Vose for advice on the use of the
SEM, and an anonymous reviewer for extremely helpful comments.
REFERENCES
Baker SC, Robinson-Beers K, Villanueva JM, Gaiser JC, Gasser CS.
1997. Interactions among genes regulating ovule development in
Arabidopsis thaliana. Genetics 145:1109–1124.
236
KRIZEK
Bechtold N, Ellis J, Pelletier G. 1993. In planta Agrobacterium
mediated gene transfer by infiltration of adult Arabidopsis thaliana
plants. CR Acad Sci 316:1194–1199.
Bowman JL, Smyth DR, Meyerowitz EM. 1991. Genetic interactions
among floral homeotic genes of Arabidopsis. Development 112:1–20.
Buttner M, Singh KB. 1997. Arabidopsis thaliana ethylene-responsive
element binding protein (AtEBP), an ethylene-inducible, GCC box
DNA-binding protein interacts with an ocs element binding protein.
Proc Natl Acad Sci USA 94:5961–5966.
Cleland RE. 1987. Auxin and cell elongation. In: Davis PJ, editor.
Plant hormones and their role in plant growth and development.
The Netherlands: Kluwer. p 132–148.
Coen ES, Meyerowitz EM. 1991. The war of the whorls: genetic
interactions controlling flower development. Nature 353:31–37.
Coen ES, Romero JM, Doyle S, Elliott R, Murphy G, Carpenter R.
1990. Floricaula: a homeotic gene required for flower development
in Antirrhinum majus. Cell 63:1311–1322.
Depicker A, van Montagu M. 1997. Post-transcriptional gene silencing
in plants. Curr Opin Cell Biol 9:373–382.
Elliott RC, Betzner AS, Huttner E, Oakes MP, Tucker WQJ, Gerentes
D, Perez P, Smyth DR. 1996. AINTEGUMENTA, an APETALA2like gene of Arabidopsis with pleiotropic roles in ovule development
and floral organ growth. Plant Cell 8:155–168.
Fobert PR, Coen ES, Murphy GJP, Doonan J. 1994. Patterns of cell
division revealed by transcriptional regulation of genes during the
cell cycle in plants. EMBO J 13:616–624.
Gaiser JC, Robinson-Beers K, Gasser CS. 1995. The Arabidopsis
SUPERMAN gene mediates asymmetric growth of the outer integument of ovules. Plant Cell 7:333–345.
Hanzawa Y, Takahashi T, Komeda Y. 1997. ACL5: an Arabidopsis gene
required for internodal elongation after flowering. Plant J 12:863–
874.
Heslop-Harrison J, Heslop-Harrison Y. 1970. Evaluation of pollen
viability by enzymatically induced fluorescence: intracellular hydrolysis of fluorescein diacetate. Stain Tech 45:115–120.
Hooley R. 1996. Plant steroid hormones emerge from the dark. Trends
Genet 12:281–283.
Jacobson DR, Moscovits T. 1991. Rapid, nonradioactive screening for
activating ras oncogenic mutations using PCR primer-introduced
restriction analysis (PCR-PIRA). PCR Methods Appl 1:146–148.
Kagaya Y, Ohmiya K, Hattori T. 1999. RAV1, a novel DNA-binding
protein, binds to bipartite recognition sequence through two distinct
DNA-binding domains uniquely found in higher plants. Nucleic
Acids Res 27:470–478.
Kim Q-T, Tsukaya H, Uchimiya H. 1998. The CURLY LEAF gene
controls both division and elongation of cells during the expansion of
the leaf blade in Arabidopsis thaliana. Planta 206:175–183.
Klimyuk VI, Carroll BJ, Thomas CM, Jones JDG. 1993. Alkali
treatment for rapid preparation of plant material for reliable PCR
analysis. Plant J 3:493–494.
Klucher KM, Chow H, Reiser L, Fischer RL. 1996. The AINTEGUMENTA gene of Arabidopsis required for ovule and female gametophyte development is related to the floral homeotic gene APETALA2.
Plant Cell 8:137–153.
Krizek BA, Meyerowitz EM. 1996. Mapping the protein regions
responsible for the functional specificities of the Arabidopsis MADS
domain organ-identity proteins. Proc Natl Acad Sci USA 93:4063–
4070.
Liu Q, Kasuga M, Sakuma Y, Abe H, Miura S, Yamaguchi-Shinozaki K,
Shinozaki K. 1998. Two transcription factors, DREB1 and DREB2,
with an EREBP/AP2 DNA binding domain separate two cellular
signal transduction pathways in drought- and low-temperature
responsive gene expression, respectively, in Arabidopsis. Plant Cell
10:1391–1406.
Ma H. 1994. The unfolding drama of flower development: recent
results from genetic and molecular analyses. Genes Dev 8:745–756.
Marzluff WF, Pandey NB. 1988. Multiple regulatory steps control
histone mRNA concentrations. Trends Biochem Sci 13:49–52.
Métraux J-P. 1987. Gibberellins and plant cell elongation. In: Davis
PJ, editor. Plant hormones and their roles in plant growth and
development. The Netherlands: Kluwer. p 296–317.
Meyerowitz EM. 1997. Genetic control of cell division patterns in
developing plants. Cell 88:299–308.
Mitchison JM, Novak B, Sveiezer A. 1997. Size control in the cell cycle.
Cell Biol Int 21:461–463.
Müller A. 1961. Zur characterisierung der Blüten und Infloreszenzen
von Arabidopsis thaliana (L.) Heynh. Kulturpflanze 9:364–393.
Nakayama T, Iwabuchi M. 1993. Regulation of wheat histone gene
expression. Crit Rev Plant Sci 12:97–110.
Ohme-Takagi M, Shinshi H. 1995. Ethylene-inducible DNA binding
proteins that interact with an ethylene-responsive element. Plant
Cell 7:173–182.
Okamuro JK, Caster B, Villarroel R, Montagu MV, Jofuku KD. 1997.
The AP2 domain of APETALA2 defines a large new family of DNA
binding proteins in Arabidopsis. Proc Natl Acad Sci USA 94:7076–
7081.
Regan SM, Moffatt BA. 1990. Cytochemical analysis of pollen development in wild-type Arabidopsis and a male-sterile mutant. Plant Cell
2:877–889.
Reiser L, Fischer RL. 1993. The ovule and the embryo sac. Plant Cell
5:1291–1301.
Riechmann JL, Meyerowitz EM. 1998. The AP2/EREBP family of
plant transcription factors. Biol Chem 379:633–646.
Robinson-Beers, Kay P, E. R, Gasser CS. 1992. Ovule development in
wild-type Arabidopsis and two female-sterile mutants. Plant Cell
4:1237–1249.
Running MP, Clark SE, Meyerowitz EM. 1995. Confocal microscopy of
the shoot apex. In: Galbraith DW, Bohnert HJ, Bourque DP, editors.
Methods in cell biology’’ San Diego: Academic Press. p 217–229.
Sablowski RWM, Meyerowitz EM. 1998. A homolog of NO APICAL
MERISTEM is an immediate target of the floral homeotic genes
APETALA3/PISTILLATA. Cell 92:93–103.
Sakai H, Medrano LJ, Meyerowitz EM. 1995. Role of SUPERMAN in
maintaining Arabidopsis floral whorl boundaries. Nature 378:199–
203.
Schneitz K, Hulskamp M, Kopczak SD, Pruitt RE. 1997. Dissection of
sexual organ ontogenesis: a genetic analysis of ovule development in
Arabidopsis thaliana. Development 124:1367–1376.
Schneitz K, Hulskamp M, Pruitt RE. 1995. Wild-type ovule development in Arabidopsis thaliana: a light microscopic study of cleared
whole-mount tissue. Plant J 7:731–749.
Sessions A, Yanofsky MF, Weigel D. 1998. Patterning the floral
meristem. Semin Cell Dev Biol 9:221–226.
Smyth DR, Bowman JL, Meyerowitz EM. 1990. Early flower development in Arabidopsis. Plant Cell 2:755–767.
Stockinger EJ, Gilmour SJ, Thomashow MF. 1997. Arabidopsis
thaliana CBF1 encodes an AP2 domain-containing transcriptional
activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low
temperature and water deficit. Proc Natl Acad Sci USA 94:1035–
1040.
Takahashi T, Gasch A, Mishizawa N, Chua N-H. 1995. The DIMINUTO
gene of Arabidopsis is involved in regulating cell elongation. Genes
Dev 9:97–107.
Talbert PB, Adler HT, Parks DW, Comai L. 1995. The REVOLUTA
gene is necessary for apical meristem development and for initiating
cell divisions in the leaves and stems of Arabidopsis thaliana.
Development 121:2723–2735.
Vergani P, Morandinin P, Soave C. 1997. Complementation of a yeast
Dpkc1 mutant by the Arabidopsis protein ANT. FEBS Lett 400:243–
246.
Weigel D, Meyerowitz E. 1994. The ABCs of floral homeotic genes. Cell
78:203–209.
Wilson K, Long D, Swinburne J, Coupland G. 1996. A dissociation
insertion causes a semidominant mutation that increases expression of TINY, an Arabidopsis gene related to APETALA2. Plant Cell
8:659–671.
Zhou J, Tang X, Martin GB. 1997. The Pto kinase conferring resistance
to tomato bacterial speck disease interacts with proteins that bind a
cis-element of pathogenesis-related genes. EMBO J 16:3207–3218.