Development 121, 2887-2895 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
2887
Genetic ablation of petal and stamen primordia to elucidate cell interactions
during floral development
Christopher D. Day, Bernard F. C. Galgoci* and Vivian F. Irish†
Department of Biology, Osborn Memorial Laboratories, P.O. Box 208104, Yale University, New Haven, CT 06520-8104, USA
*Present address: Woods’ Edge Farm, 532 Stanek Road, Muscoda, WI 53573, USA
†Author for correspondence
SUMMARY
Two models have been proposed to explain the coordinated
development of the four whorls of floral organs. The spatial
model predicts that positional information defines the four
whorls simultaneously, and that individual organs develop
independently of surrounding tissues. The sequential
model suggests that inductive events between the outer and
inner whorl primordia are required for appropriate
organogenesis. To test these models we have genetically
ablated second and third whorl floral organ primordia to
determine if organ identity, number or position are
perturbed in the first or fourth whorls. We used diphtheria toxin to specifically ablate floral cells early in development in Nicotiana tabacum and in Arabidopsis thaliana.
Second and third whorl expression of the diphtheria toxin
INTRODUCTION
Floral organ primordia arise from the coordinated divisions of
cells on the flanks of the florally determined shoot apical
meristem. In response to floral inductive signals, the meristematic apex generally becomes more domed, and increases in
mitotic activity (Steeves and Sussex, 1989). The first whorl of
floral organ primordia arising from the dividing cells on the
flank of the apex gives rise to sepals. Generally, the subsequent
organ primordia initiate from outer to inner whorls, and differentiate as petals, stamens and carpels.
It appears that the mechanisms specifying floral organ identities are distinct from those specifying number and position of
floral organ primordia. Floral homeotic mutants in Antirrhinum
and Arabidopsis alter the identity of organs, but do not
generally perturb the number or position of organ primordia
within a whorl (Meyerowitz et al., 1991). Other floral
mutations disrupt organ number, but otherwise have no
obvious effect on floral development (Clark et al., 1993).
There are two models that attempt to describe how the cells
of the floral meristem interact to initiate organ development.
Holder (1979) proposed that positional signals are laid down
simultaneously in the meristem, and that floral organs then differentiate independently of one another. In contrast, Wardlaw
and others (Wardlaw, 1957; Heslop-Harrison, 1964; Hicks and
Sussex, 1971) suggested that sequential inductive signals were
A chain coding sequence (DTA) was conferred by the Arabidopsis APETALA3 (AP3) promoter. Both Nicotiana and
Arabidopsis flowers that express the AP3-DTA construct
lack petals and stamens; it appears that the second and
third whorl cells expressing this construct arrest early in
floral development. These results show that first and fourth
whorl development is normal and can proceed without
information from adjacent second and third whorl
primordia. We propose that positional information
specifies the establishment of all four whorls of organs
prior to the expression of AP3 in the floral meristem.
Key words: Arabidopsis, Nicotiana, diphtheria toxin, ablation,
pattern formation
required to specify the floral pattern. Inductive signals from the
outer (first) whorl were proposed to trigger the initiation of the
second whorl; development of the second whorl allowed the
production of a second signal which induced development of
the third whorl, and so on. An important difference between
this sequential model and the spatial model is that development
of a particular organ in the sequential model is dependent on
signals from the previous whorl.
A number of surgical experiments have been performed on
floral meristems in an effort to distinguish between these
models. Most of these surgical experiments have been interpreted as supporting a sequential model. Excisions of individual whorls were shown to affect the normal development of
Nicotiana flowers grown in sterile culture (McHughen, 1980).
Removal of stamen primordia significantly inhibited carpel
development and was presented as evidence to support the
sequential model. Floral meristems of different species have
been bisected and all showed the potential to regenerate two
flowers if the bisection was performed prior to the initiation of
sepal primordia (Cusick, 1956; Hicks and Sussex, 1971;
Soetiarto and Ball, 1969). These observations showed that cells
in the floral meristem were not irreversibly fated to give rise to
a particular organ. Bisections of floral meristems at successive
stages indicated that the developmental potential of regenerating flower buds became limited with time. For example, if
meristem bisections were performed after sepal primordia are
2888 C. D. Day, B. F. C. Galgoci and Vivian F. Irish
initiated, no sepals regenerated on the operated side of the apex.
Such information suggested that a developing flower had a
series of organ initiation phases. Based on these observations,
Hicks and Sussex (1971) proposed a refined version of the
sequential model. Since a subtending whorl (e.g., sepals) was
not required on the operated side of the apex to initiate the next
whorl (e.g., petals) and the pattern of regeneration was
organised around the new apex, they suggested that the
inductive signal was not derived from the extant organ
primordia but from the center of the floral apex. While these
surgical experiments have been useful in studying cell interactions in the floral meristem, it is impossible to reliably remove
all the cells of an organ primordium. In addition, other cells are
damaged during the manipulation of the floral meristem.
More efficient ways of removing cells with the smallest
possible impact to the system have superseded traditional
surgical techniques. Lasers have been used to specifically kill
individual cells in Caenorhabditis elegans (Kimble, 1981;
Sulston and White, 1980) as well as in plants (Croxdale et al.,
1992). Another useful technique is genetic cell ablation (Bellen
et al., 1992; Breitman et al., 1987; Palmiter et al., 1987). The
success of this technique relies on the use of a tissue-specific
promoter to express a cell-autonomous toxic gene product in
the cells to be removed. This approach has been used successfully in plants using the toxic products of the diphtheria toxin
A chain gene (DTA) (Thorsness et al., 1993) or the barnase
gene (Mariani et al., 1990; Goldman et al., 1994). The DTA
toxin is cell-autonomous since without the diphtheria toxin B
chain and extracellular targetting sequences, it cannot be transported across the plasma membrane (Pappenheimer, 1977).
DTA functions by ribosylating the EF2 translation initiation
factor and consequently inhibiting all protein translation
(Collier, 1975). Cells expressing DTA are unable to maintain
protein synthesis, cannot divide, and eventually die.
The experimental approach we have taken is to use genetic
ablation with DTA to remove the second and third whorl cells
in the floral meristem at an early stage in development. The
removal of two whorls from the floral meristem allows us to
make predictions about organ formation based on the two
models of floral development. If the spatial model is correct,
we would expect the first whorl of sepals and the fourth whorl
carpels to develop normally. Conversely, the sequential model
would predict disruptions in fourth whorl and/or first whorl
development. We used the Arabidopsis APETALA 3 (AP3)
promoter (Irish and Yamamoto, 1995) to drive the expression
of the DTA in second and third whorl primordia in Arabidopsis thaliana and Nicotiana tabacum floral meristems. In both
species the ablated flowers failed to develop petals and
stamens. Carpel and sepal development in both species,
however, appeared to be largely unaffected. We propose that
spatial signals defining the organ primordia are established
before AP3 is expressed in the floral meristem. We discuss
these results in the context of models of floral development.
MATERIALS AND METHODS
Plant material
Nicotiana tabacum cv SR1 plants were grown at 28°C under standard
greenhouse conditions. Arabidopsis thaliana ecotype NoO were
grown in a growth room at 22-24°C under a 16 hour day/8 hour night
regime, with lights at 175 µmol m−2 sec−1 at the pot top.
Strains and molecular cloning
Escherichia coli strain DH5α was used to grow plasmids by standard
procedures (Sambrook et al., 1989). Agrobacterium tumefaciens strain
LBA4404 (Clonetech Laboratories, Inc., Palo Alto, CA) was used for
plant transformation.
The chimeric AP3-DTA contruct was made by subcloning the DTA
PstI, SacI fragment from pSKDT-A (Bellen et al., 1992) into pBI221
(Clonetech Laboratories, Inc., Palo Alto, CA) to generate plasmid
pBIDN. The DTA-NOS HindIII, EcoRI fragment was subcloned into
pBluescript KS+ to generate pKSDN. The 1.9 kb HindIII genomic
fragment including 1.7 kb of AP3 5′ sequence and the first 159 bp of
the AP3 gene were subcloned into pKSDN to generate a translational
fusion (pKSADN). The translational fusion, XhoI, EcoRI fragment
from pKSADN, was then subcloned into the pBI101 binary vector
(Jefferson, 1986), which was digested with SalI and EcoRI, to give
pBIADN. The binary vector construct, pBIADN, was transferred into
Agrobacterium by electroporation (Wen-jun and Forde, 1989).
Plant transformation
Transgenic Nicotiana plants were produced by leaf disc transformation (Horsch et al., 1988) using Agrobacterium transformed with
pBIADN. Transgenic Arabidopsis plants were produced by root transformation using a similar protocol to that of Valvekens et al. (1988).
Transgenic plants were selected by their kanamycin resistance. Transgenic Arabidopsis lines were all primary transformants since the
transgene could not be transmitted to the next generation.
Histology and microscopy
Tissue was fixed using FAA (3.7% formaldehyde, 50% ethanol, 5%
acetic acid) by vacuum infiltration for 30 minutes and dehydrated in
a graded ethanol series. Nicotiana flowers were embedded in paraffin
and cut into 8 µm sections. Sections were hydrated and stained in
DAPI, 1 µg ml−1, for 30 minutes and visualised with fluorescence
optics. Arabidopsis flowers were embedded in SPURRS resin and cut
into 2 µm sections, then stained with a solution of 0.05% toluidine
blue, 1% sodium borate.
For scanning electron microscopy, dehydrated flowers were critical
point dried in liquid CO2. Specimens were sputter coated with gold
paladium and examined in an ISI SS40 scanning electron microscope
with an accelerating voltage of 10 kV.
In situ hybridisation
Preparation of the Nicotiana tissue for in situ hybridisation was
carried out as described by Jackson (1991). An antisense RNA probe
of NAG1 was prepared from the pKY60 plasmid (Kempin et al.,
1993). The plasmid was linearised with BglII, which removed the
MADS box region and was transcribed with T7 polymerase
(Boehringer Mannheim Corp., Indianapolis, IN). RNA probes were
labelled with digoxigenin-UTP (Boehringer Mannheim Corp., Indianapolis, IN) according to the manufacturer’s instructions. 8 µm
paraffin sections of transgenic Nicotiana buds were affixed to ProbeOn Plus slides (Fisher Scientific, Pittsburgh, PA). Pretreatment of
slides, hybridisation, and washing were carried out according to the
method of Langdale et al. (1988) and (Jackson, 1991) with slight modifications. The digoxigenin label was immunologically detected using
anti-digoxigenin-alkaline phosphatase, and nitroblue tetrazolium and
X-phosphate to detect alkaline phosphatase activity according to the
manufacturer’s instructions (Boehringer Mannheim Corp., Indianapolis, IN).
RESULTS
Construction of a chimeric DTA toxin
A prerequisite for the success of this experiment is a highly
tissue-specific promoter which restricts the expression of the
toxin to cells in the second and third whorl. We chose to use
Genetic ablation of petal and stamen primordia 2889
NOS
AP3 promoter
NKLACLQ V LAMDP
500 bp
AP3
DTA
Fig. 1. Schematic diagram of the AP3-DTA chimeric toxin. The
white box represents AP3 sequence; the shaded box represents DTA
sequence. Hatched line indicates the nopaline synthase transcription
terminator sequence. The amino acid sequence of the translational
fusion between AP3 and DTA is indicated below the diagram.
the promoter of the Arabidopsis AP3 gene, which is expressed
in petal and stamen primordia (Jack et al., 1992). AP3 transcripts can first be detected at stage 3
(stages according to Smyth et al., 1990)
in eight to ten cells in all three cell layers
of the floral meristem.
We used a 1.9 kb AP3 genomic clone
to construct a translational fusion
between the AP3 promoter and the DTA
coding region (Fig. 1). The genomic
clone included 1.7 kb of 5′ sequence,
which is sufficient to confer normal AP3
expression in Arabidopsis (Irish and
Yamamoto, 1995), and the first 162 bp
of the AP3 open reading frame.
Expression of the chimeric AP3DTA toxin in Arabidopsis
specifically ablates second and
third whorl organs
The primary transgenic Arabidopsis
plants that expressed the chimeric DTA
protein had a striking phenotype which
consisted of a normal flower with no
Fig. 2. Developmental stages of wild-type
and transgenic Arabidopsis flowers.
Comparisons of wild-type (A,C,E,G,I) with
transgenic Arabidopsis flowers expressing
the chimeric AP3-DTA contruct
(B,D,F,H,J). (A) A mature wild-type flower.
(B) The transgenic Arabidopsis flower
(black arrow indicates a sepal); two first
whorl sepals have been removed (white
arrow indicates the scar) to reveal the
gynoecium in the transgenic flower. (C) A
longitudinal section of a stage 11 wild-type
flower. (D) A transgenic Arabidopsis flower
at a similar stage to (C). (E,F) Longitudinal
sections through the gynoecium at late stage
12; compare ovule development between (E)
wild-type and (F) the transgenic
Arabidopsis. In F some ovules did not fully
develop integuments. (G,H) Longitudinal
sections through flowers at stage 3 were
similar for (G) wild-type and (H) the
transgenic Arabidopsis. (I,J) By stage 6 the
organ primordia were clearly visible in wildtype (I) and absent in the transgenic (J)
Arabidopsis. Scale bars, C,D, 100 µm; E,F,
I,J, 50 µm; G,H, 25 µm.
petals or stamens (Fig. 2). Southern analysis of one line, with
probes from the right and left border of the T-DNA insert,
showed that there were up to five copies of the chimeric gene
inserted at three locations in the genome (data not shown). Five
other independent transgenic lines were obtained which
displayed similar phenotypes.
The transgenic Arabidopsis flowers were sectioned to
determine the morphology at various stages of development.
The mature transgenic flowers had two distinct whorls of
organs which were almost identical to those found in the first
and fourth whorls of the wild type (Fig. 2C,D). The first whorl
contained four sepals which appeared normal at the morphological and histological level. The remaining organs consisted
of two fused carpels which appeared normal, although the
2890 C. D. Day, B. F. C. Galgoci and Vivian F. Irish
ovules occasionally displayed an
aberrant structure which is discussed
below. The epidermal cell layer of the
sepals and the gynoecium are adjacent
and there is no apparent scar tissue from
the absent second and third whorls (Fig.
2D). From the phenotype of these transgenic lines we conclude that in Arabidopsis two whorls, the second and
third in wild-type flowers, have been
specifically ablated and that this
ablation does not cause any significant
defect in the development of the first or
fourth whorls.
Second and third whorl cells are
ablated early in development
AP3 transcripts are first detectable at
the stage when sepal primordia are just
beginning to initiate (Jack et al., 1992).
We sectioned flower buds to determine
when we could detect the onset of morphological anomalies. At stage 3 there
are no obvious differences between
wild-type and transgenic flowers (Fig.
2G,H). By stage 6, wild-type flowers
had apparent stamen and petal
primordia (Fig. 2I). A section from the
equivalent stage in the transgenic
flower bud lacked petal and stamen
primordia (Fig. 2J). This implies that
the cells that are normally determined
to become organs in the second and
third whorls are ablated early in development. Later, at stage 7, after the
carpels have started to initiate, the wildtype flower has a distinct pattern of four
whorls of organs. At a similar stage, the
transgenic flowers had only two whorls
of organs. In addition, sepal and carpel
epidermal cells were in contact at the
base of the organs (data not shown). If
inductive signals are exchanged
between these two whorls they do not
alter organ development since these
primordia develop into essentially
normal organs.
Ovule development is aberrant in
transgenic Arabidopsis flowers
In wild-type carpels an outer and inner
integument grow to surround the ovule
(Robinson-Beers et al., 1992). In the
transgenic
Arabidopsis
flowers,
however, some of the ovules did not
appear to have normal integuments
(Fig. 2F). This phenotype may be due
to specific cell ablation or to an
inductive effect due to the loss of petals
and stamens. Specific cell ablation is
more consistent with the known
Fig. 3. Genetic ablation of the petals and stamens in two different transgenic lines of
Nicotiana expressing the chimeric AP3-DTA contruct. (A) Wild type, longitudinally split to
show inner organs. (B) Longitudinally split transgenic Nicotiana flower displaying the
strong phenotype has an essentially normal gynoecium despite the absence of petals and
stamens. (C) A patch of stamen-like tissue on an otherwise normal gynoecium from a
transgenic, strong phenotype, Nicotiana. (D) Wild-type gynoecium (far left) compared with
typical gynoecium from the transgenic, strong phenotype, Nicotiana with the outer organs
(sepals, petals and stamens for the wild type; sepals for the transgenic Nicotiana) removed
for clarity. Of the flowers that had carpels, 15% had a small mosaic sector which caused
some distortion of the gynoecium (second from left). 23% had more severe distortions (third
from left), while 18% had up to two mosaic sectors which resulted in severe distortion of the
carpels and an inhibition of the style fusion and elongation (fourth from left). 44% of the
gynoecia were essentially normal, similar to that in B. (E) Representative flowers from
transgenic, intermediate phenotypes. The severity of the phenotype increases from left to
right.
Genetic ablation of petal and stamen primordia 2891
expression pattern of AP3, since AP3 is expressed late in pistil
development at the base of the integument (Jack et al., 1992).
Not all of the integuments were ablated in the transgenic
flowers. This partial effect may be due to weak AP3 expression
in this tissue, which lowers the amount of chimeric toxin below
a threshold required for toxicity. While in other systems the
DTA toxin has proved to be very potent (Yamaizumi et al.,
1978), either the construct or the position of the transgene
insert may have provided a low level of DTA toxin, resulting
in a threshold effect. The existence of such a toxic threshold
was also suggested by our studies with transgenic Nicotiana.
Therefore, the loss of some ovule integuments was likely to be
due to specific cell ablation rather than to an inductive effect
resulting from petal and stamen ablation.
When the transgenic Arabidopsis flowers were pollinated
with wild-type (Landsberg erecta) pollen some seed developed
to maturity. None of the seed we recovered were kanamycin
resistant, which suggested that the AP3-DTA construct was
also expressed gametophytically or in the embryo.
Expression of the chimeric AP3-DTA toxin results in
loss of second and third whorls in Nicotiana
We also used the AP3-DTA construct to transform Nicotiana
tabacum. The primary transformants of Nicotiana we
generated had a variety of phenotypes which appeared to
depend on the copy number of T-DNA inserts in the genome.
As with Arabidopsis, the inserts were characterised by
Southern blot analysis using right and left border probes
Fig. 4. Delayed development of
the gynoecium in the transgenic,
strong phenotype, Nicotiana.
Development of wild-type
Nicotiana (A-C) compared with
transgenic, strong phenotype,
Nicotiana (D-F) flowers. Scale
bars, 100 µm.
specific for the T-DNA insert (data not shown). The strongest
phenotype correlated with three inserts; an intermediate
phenotype with two transgene inserts; and weak phenotypes
appeared in two lines containing one insert each.
In the transgenic Nicotiana line with the strong phenotype,
the petals and stamens are absent and the phenotype is quite
similar to that seen with Arabidopsis (Fig. 3B). The first whorl
has an essentially normal calyx (fused sepals) with an inner
whorl of carpels which are fertile when pollinated with wildtype pollen. The calyx occasionally contains four fused sepals,
as compared to five in wild type, and the mature sepals are
usually slightly longer (Fig. 3B). No petals or stamens can be
detected. Unlike transgenic flowers from Arabidopsis,
however, carpel development in the transgenic Nicotiana
flowers is delayed compared to wild type (Fig. 4). Although
the outgrowth of the carpels is significantly delayed, eventually these carpels develop to full size. The transgenic Nicotiana
flowers also occasionally displayed defects in the final morphology of the gynoecium. Occasionally, what appeared to be
mosaic patches of petal or stamen tissue developed on the
carpels (Fig. 3C). One explanation for the appearance of these
patches of petaloid or stamenoid tissue is that expression of the
chimeric toxin is below a required threshold level in these cells.
In addition, the gynoecia produced in this transgenic line were
occasionally kinked or wrinkled (Fig. 3D).
We have shown that carpel development in transgenic
Nicotiana with the strong phenotype is delayed (Fig. 4). This
may be due to the delay or loss of an inductive signal to activate
2892 C. D. Day, B. F. C. Galgoci and Vivian F. Irish
the organ identity genes in the fourth whorl. NAG1 is specifically expressed in the third and fourth whorls of wild-type
floral meristems and is required for organ identity and determination in the floral meristem (Kempin et al., 1993). We used
mRNA in situ analysis to determine if NAG1 is expressed in
the retarded fourth whorl primordia of transgenic Nicotiana
buds displaying the strong phenotype. We find that the NAG1
transcript is expressed in the fourth whorl during the period
that the carpel growth is delayed (Fig. 5C). Expression of
NAG1 in the carpels at this stage suggests that the delay in
carpel development is not due to the loss of an inductive signal
from the petals or stamens.
The transgenic Nicotiana line that displayed the intermediate phenotype showed flower phenotypes ranging from almost
wild type, to having only one or two petals and stamens (Fig.
3E). The number of petal and stamen primordia that developed
was variable and the organ fusions appeared disorganised. The
petals were often misfused at their margins allowing petalloid
segments to develop outside the corolla. When a petal was
absent, fusion still occurred at the base of the corolla but rarely
continued into the petal lobe, resulting in a slit corolla with one
to four petals. Occasionally we have seen a complete corolla
form from the fusion of only four petals. We rarely observed
half petals. The temporal development of the gynoecium was
similar to wild type in these flowers.
The transgenic Nicotiana plants that displayed a weak
phenotype generally had wild-type flowers. Occasionally,
abnormal fusions of the corolla were observed.
Cell death induced by the transgene can be followed
using DAPI
One way to visualise cell death is to look for degradation of
chromosomal DNA. In mammalian cells, DTA is one of the
inhibitors of protein synthesis, which can cause characteristic
internucleosomal fragmentation of DNA (Kochi and Collier,
1993; Martin, 1993). To determine if we can detect apoptoticlike cell death we stained young Nicotiana floral buds with
DAPI to visualise DNA. Wild-type Nicotiana buds display a
characteristic pattern of nuclear DAPI fluorescence (data not
shown). Transgenic Nicotiana buds displayed much brighter
DAPI fluorescence in cells between the first and fourth whorl
(Fig. 5A,B). In addition, this fluorescence appeared to be distributed throughout the cytoplasm. Similar bright, diffuse
DAPI fluorescence has been observed in cells undergoing
apoptotic cell death (Kulkarni and McCulloch, 1994). We
interpret the pattern of fluorescence we see as resulting from
cells that are dying due to expression of DTA.
DISCUSSION
Loss of petal and stamen primordia has little effect
on the development of other floral organs
The spatial model predicts that positional information specifying the floral pattern is set early in development and implies
that the development of an organ is independent of the surrounding tissues. The phenotype of the transgenic flowers we
have generated in Arabidopsis correlates with these predictions
since sepal and carpel development were essentially unaffected
by the removal of petal and stamen primordia. Although the
transgenic Nicotiana plants displaying the strong phenotype
occasionally showed a variety of defects in carpel development, the fact that these plants frequently produce a normal
and functional gynoecium indicated that information from the
second and third whorl primordia was not necessary for carpel
development. We have shown that cell ablation occurs early,
just after sepal primordia become apparent, in both Arabidopsis and Nicotiana (Figs 2I,J, 4). In these ablated flowers, the
number and position of first and fourth whorl organs are
normal. Since essentially normal first and fourth whorl development is observed in both Nicotiana and Arabidopsis, we
propose that growth centers defining the position of the organ
primordia in all four whorls are set up before the expression of
the chimeric toxin in the second and third whorls. This positional information is sufficient to autonomously direct the
development of floral organ primordia, consistent with the predictions of the spatial model.
Analysis of floral homeotic mutants in several systems has
shown that the specification of organ identity in a particular
whorl is not dependent on the identity of the previous whorl
(Meyerowitz et al., 1991; Schwarz-Sommer et al., 1990; van
der Krol and Chua, 1993). Therefore, the specification of organ
identity has previously been considered to be consistent with
a spatial model (Bowman et al., 1991; Haughn and Somerville,
1988). Since these homeotic mutants do not generally disrupt
organ number or position, it appears that the mechanisms specifying floral organ identity are distinct from those specifying
organ number and position (Meyerowitz et al., 1991). Crone
and Lord (1994) observed that organ primordia in floral
homeotic mutants of Arabidopsis initiate in a normal manner,
Fig. 5. Characterisation of cell death and fourth whorl gene expression in the transgenic, strong phenotype, Nicotiana. (A) Light micrograph of
longitudinal section. (B) DAPI staining of section in A visualised with fluorescence optics. The cells located between the first and fourth whorls
are much brighter than background with fluorescence appearing in the cytoplasm. (C) A similar section to that in A which has been probed with
digoxigenin-labelled NAG1 antisense RNA, which shows expression of NAG1 in the retarded fourth whorl. Scale bar, 100 µm.
Genetic ablation of petal and stamen primordia 2893
and only subsequently show deviations in cell number and
division patterns from wild type. Therefore, the organ identity
genes are probably not required for the initiation of organogenesis. A mechanism establishing organ identity as distinct
from that of establishing floral organ position and number can
also be inferred from the phenomenon of floral reversion.
Impatiens balsamina flowers may revert to vegetative growth
if the floral stimulus is removed but the number and position
of newly developing vegetative primordia remain typical of the
floral meristem (Battey and Lyndon, 1984, 1990). Consequently, the development of the organ pattern might be
dependent on neighbouring whorls to coordinate the organ
positions within the meristem by biochemical (Wardlaw, 1949;
Young, 1978) or biophysical signals (Green, 1988). However,
our observations suggest that positional coordinates are established in the floral meristem by the stage of sepal primordium
initiation.
Although positional coordinates appear to be defined at an
early stage of floral development, inductive interactions
between cells in different regions of the meristem may occur
at an earlier stage. Several genes are expressed in the meristem
prior to the initiation of sepal primordia and may have a role
in establishing these positional cues. These genes include floricaula and fimbriata in Antirrhinum, and LEAFY in Arabidopsis (Coen et al., 1990; Simon et al., 1994; Weigel et al., 1992).
Mutations in all three of these genes affect the floral phyllotaxy, and so may be required to establish the position of floral
organ primordia.
Inter-whorl signals may cause some abnormalities
in Nicotiana transgenic flowers
Fourth whorl development in Nicotiana transgenic flowers was
retarded, but eventually normal pistils could be observed (Fig.
3B). We suggest that this delay is not due to the loss of an
inductive signal, since we observe expression of NAG1 in the
fourth whorl region (Fig. 5C). One possibility to explain this
retarded growth could be the loss of general growth regulators.
Growth regulators such as hormones could be acting to
promote carpel outgrowth. Stamens are known to be a source
of gibberellins (Weiss et al., 1990; Kinet et al., 1985), and may
play a role in regulating the pace of carpel development. In the
transgenic Nicotiana flowers the loss of such growth regulators may result in the delay and distortion of carpel development. In this scenario, hormones would not be required to
specify organ position and number, but would be more general
in their action to promote growth. This effect may be more pronounced in Nicotiana, due to the length of time required for
floral organogenesis in this species. Since no Nicotiana
mutants affecting stamen development have been described,
this hypothesis cannot be tested using mutants. In Antirrhinum,
a close relative of Nicotiana, homeotic mutants deficiens and
globosa result in stamens that are transformed to carpelloid
organs (Sommer et al., 1990; Trobner et al., 1992). In these
mutants it is not unusual to have a fused third whorl gynoecium
with an absent or reduced fourth whorl. It is not clear if this
phenotype is due to a similar delay of fourth whorl development or to other factors.
A proportion (56%) of the carpels in the strong transgenic
Nicotiana phenotype display mosaic patches of petal or stamen
tissue. These patches could be due to an inductive signal
emanating from the first whorl primordia, which are physically
A. Wild type Nicotiana meristem
Side view
Differentiated flower
Top view
B. Bisected meristem
C. Ablated meristem
Fig. 6. A model which accounts for the observations of the surgical
bisection and genetic ablation experiments. (A) Wild-type
development of a Nicotiana flower. The first two columns represent a
schematic side and top view of the meristem at an early stage in
floral development. Arrows represent putative intra-whorl signals. At
the stage depicted, the sepal whorl no longer expresses the intrawhorl signal. The duration of the intra-whorl signal in other whorls is
illustrated by the thickness of the arrow; light arrows indicate that the
signalling process is nearly complete, while heavier arrows indicate
that the signal will be active for longer periods. The last column
represents a schematic top view of a differentiated flower, with five
sepals (grey), five petals (orange), five stamens (pink), and two
carpels (blue). (B) Schematic representation of signals resulting in
regeneration in a surgically bisected meristem. Inductive signals
within a whorl can account for the regeneration of the cells at the
operated side of the apex. Based on the relative duration, these
putative signals could be sequentially restricted to whorls, resulting
in complete regeneration within the fourth whorl, but only partial
regeneration in the third and second whorls, with no regeneration in
the first whorl. (C) Genetic ablation of an entire whorl results in the
loss of intra-whorl signals, but does not affect first or fourth whorl
development. (B,C). Coloured organs represent development of that
organ, as in wild type. A white organ in a whorl indicates that the
particular organ does not always develop. A pale colour indicates a
position in the floral meristem which does not initiate organ
primordia.
apposed to the fourth whorl cells, such that a signal originally
destined to induce second or third whorl development would
now act on fourth whorl cells. However, such an inductive
signal does not appear to affect all fourth whorl cells, since
many of the flowers derived from the strong transgenic line
show normal carpel morphology. Such an inductive signal may
act late in development, as the fourth whorl cells are differentiating.
An alternative hypothesis to explain the mosaic patches is
that some of the second or third whorl cells are not killed by
the toxin if the toxicity drops below a threshold. These cells
2894 C. D. Day, B. F. C. Galgoci and Vivian F. Irish
might be recruited into the fourth whorl forming a mosaic
patch of petal or stamen tissue on the side of the gynoecium.
The number of cells recruited determines the size of the sector
until, in the case of large sectors, there is distortion of the
gynoecium (Fig. 3D). This observation suggests that the establishment of the carpel primordia may occur subsequent to cell
fate determination in these transgenic Nicotiana plants.
The chimeric diphtheria toxin has a low toxicity
Diphtheria toxin has been used successfully in several other
systems to induce cell death (Bellen et al., 1992; Breitman et
al., 1987). Only a few molecules of the toxin are sufficient to
kill mammalian cells (Yamaizumi et al., 1978). We have used
a modified toxin, with 54 amino acids of the AP3 coding
sequence fused to the amino terminal end of the DTA coding
region, which appears to have a lower toxicity. This reduced
toxicity is apparent in the phenotypic series of the different
transgenic Nicotiana lines, since the severity of the phenotypes
are proportional to copy number. Hence, there seems to be a
threshold level of chimeric toxin which must be reached before
it is lethal. Since the level of AP3 expression in ovule integuments is relatively low (Jack et al., 1992), threshold levels of
toxicity could explain why only some of the integuments are
ablated in the AP3-DTA transgenic Arabidopsis. Also, mosaic
patches of petal and stamen tissue can occasionally be observed
on the carpels in the phenotypically strong transgenic Nicotiana
flowers. A cell which differentiates as a petal or stamen cell in
these transgenic flowers should be ablated, but in the mosaic
patches the level of toxin produced may not be lethal.
Pattern formation during floral development
Our results appear to contradict the conclusions of surgical
experiments that inductive interactions between whorls play an
important role in establishing the floral pattern until relatively
late in development. We propose that the regenerative capability of a floral meristem could be sequentially restricted to individual whorls. In other words, inductive signals within a whorl
could function until quite late in development, but inductive
interactions between whorls would be restricted to a very early
phase of floral development, prior to the expression of AP3. By
this model, the results from the surgical experiments could be
explained by signals being disseminated within a whorl (Fig.
6). After bisection, only those whorls still competent to transmit
such an intra-whorl inductive signal would show regeneration
of organs. Since our genetic ablation studies result in complete
elimination of individual whorls, no such regeneration is
possible. Future experiments, aimed at ablating only part of a
whorl, will help to distinguish whether such a model is correct.
We wish to thank Dr Hugo Bellen for providing the pSKDT-A
plasmid, Dr Marty Yanofsky for the pKY60 plasmid, and Dr Steve
Dellaporta for help with photography. Also we would like to thank
members of the Irish laboratory for critical comments and useful suggestions on the manuscript. This work was supported by grants from
the NSF (no. IBN-9220536) and the USDA (no. 92-37304-7716) to
V. F. I.
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(Accepted 13 June 1995)