Download Expression of floricaula in single cell layers of

Development 121, 27-35 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
27
Expression of floricaula in single cell layers of periclinal chimeras activates
downstream homeotic genes in all layers of floral meristems
Sabine S. Hantke, Rosemary Carpenter and Enrico S. Coen
John Innes Centre, Colney Lane, Norwich NR4 7UH, UK
SUMMARY
We show that the flowering sectors on plants mutant for
floricaula (flo), a meristem identity gene in Antirrhinum
majus, are periclinal chimeras expressing flo in either the
L1, L2 or L3 cell layer. Flower morphology is almost
normal in L1 chimeras, but altered in L2 and L3 chimeras.
Expression of flo in any one cell layer results in the
expression of organ identity genes, deficiens (def) and plena
(ple) in all three cell layers of the chimeras, showing that
flo acts inductively to promote gene transcription. The acti-
vation of both def and ple is delayed, and the expression
domain of def is reduced, accounting for some of the phenotypic properties of the chimeras. Furthermore, we show
that flo exhibits some cell-autonomy with respect to
autoregulation.
INTRODUCTION
may be involved in the genetic control of meristem behaviour.
To investigate this possibility on a molecular level, it is
important to know the expression pattern of the gene under
study, both in wild type and in the chimeras. For example, is
the gene normally expressed in one layer or in several, and is
its expression pattern within a layer altered in the chimeras? It
is also necessary to determine how the activity of other genes
might be influenced in the chimeras and understand how this
could alter development.
The study of flower development provides a suitable system
for addressing these questions because many of the key developmental genes have been described. One class of genes affect
meristem identity; mutations in them cause shoots to grow in
place of flowers. Examples include the floricaula (flo) gene of
Antirrhinum (Carpenter and Coen, 1990; Coen et al., 1990) and
its homologue, leafy in Arabidopsis (Weigel et al., 1992). A
second class of genes affect organ identity; mutations in them
result in homeotic replacements of various types of floral
organ. Examples include deficiens (def) and plena (ple) of
Antirrhinum (Sommer et al., 1990; Bradley et al., 1993) and
their counterparts apetala3 and agamous in Arabidopsis (Jack
et al., 1992; Yanofsky et al., 1990). In the def mutant, petals
are replaced by sepals and stamens are replaced by carpels.
Accordingly, def is expressed in whorls 2 and 3 of wild-type
flowers (Sommer et al., 1991). In the ple mutant, stamens and
carpels are replaced by perianth organs, and the ple gene is
expressed in whorls 3 and 4 of wild-type flowers (Bradley et
al., 1993). A further class of genes, represented by fimbriata
(fim) in Antirrhinum, acts between the meristem and organ
identity genes in a sequence of gene activation events (Simon
et al., 1994). RNA in situ hybridization experiments show that
all of the genes so far analysed are expressed in all three layers
of the floral meristem within a given domain. For each gene,
Plant meristems are groups of dividing cells that give rise to
all postembryonic organs. The shoot meristems of many
angiosperms consist of three distinct cell layers: the epidermal
layer (L1), the subepidermal layer (L2), and the inner core (L3)
(Satina et al., 1940; Huala and Sussex, 1993). Cells of the L1
divide anticlinally throughout development, so that daughter
cells remain in the same layer, whereas cells of the L3 divide
in all planes. In contrast, cells of the L2 initially divide anticlinally, but can also divide periclinally during organ development. As a result of the planes of division, the three cell layers
are clonally distinct (Satina et al., 1940), but cells of all layers
participate in the formation of organs. The L1 gives rise to the
epidermal structures, the L2 gives rise to the subepidermal
mesophyll and the germ cells of the reproductive organs, and
the L3 forms most of the internal and vascular tissues of the
plant (Satina and Blakeslee, 1941 and 1943; Stewart and Burk,
1970; Dermen and Stewart, 1973).
The processes that coordinate the activities of cells in a shoot
meristem during development can be analysed by using periclinal chimeras. These are plants in which the genotype of one
layer differs from that of the other layers. In chimeras that are
generated experimentally, the affected layer is usually determined by using colour, morphological or cytological markers,
which are associated with the gene of interest (Huala and
Sussex, 1993). Most of the chimeras have been analysed phenotypically, but the underlying gene interactions have not been
studied at the molecular level (for examples, see TilneyBassett, 1986; Sinha and Hake, 1990; Stewart et al., 1972;
Szymkowiak and Sussex, 1992 and 1993; Zimmerman and
Hitchcock, 1951; Carpenter and Coen, 1994). In some cases,
the findings have suggested that interactions between layers
Key words: Antirrhinum majus, floricaula, flower development,
periclinal chimeras, homeotic genes
28
S. S. Hantke, R. Carpenter and E. S. Coen
the expression boundaries appear to pass continuously through
different layers, suggesting that expression is coordinated
between the layers, although it is unclear how this is achieved.
The interactions between floral homeotic genes both within
and between layers can be determined through the analysis of
their expression patterns in chimeras. In the accompanying
paper (Carpenter and Coen, 1994), the genetic and morphological analysis of plants chimeric for flo is described. They
arose as flowering sectors on plants carrying the unstable allele
flo-613 (Coen et al., 1990), which has a copy of the transposable element Tam3 inserted in the second exon. The genetic
behaviour of progeny obtained from flowers on the sectors
suggests that each flowering sector arose as the result of a
single Tam3 excision event early in meristem development.
The flowers observed on the sectors do not have the exact wildtype phenotype. Mainly petal and stamen morphology is
affected to various degrees, so that the phenotypes can be
divided into three different classes based on their severity: near
wild type, intermediate and extreme. The different phenotypes
are not transmitted to the progeny of these flowers, but can only
be maintained vegetatively through cuttings. Taken together,
these results strongly suggest that the flowering sectors are
chimeric for flo (Carpenter and Coen, 1994).
Here we demonstrate by in situ hybridization and DNA
analysis that the plants derived from flowering sectors are periclinal chimeras, expressing the flo gene in only one of the
meristematic cell layers: L1, L2, or L3. The flower phenotypes
of the chimeras mainly depend on the layer in which flo is
expressed; sequence analysis of excision alleles in the chimeras
reveals that imprecise excisions can lead to further phenotypic
variation. The expression patterns of organ identity genes def
and ple reveal that flo acts inductively across cell layers to
activate downstream genes.
MATERIALS AND METHODS
DNA and PCR analysis
DNA extraction and Southern blot analysis were carried out as
described in Coen et al. (1986). The flo probe used for hybridization
is derived from the 1.6 kb flo cDNA clone (Coen et al., 1990). Polymerase chain reactions were carried out under standard buffer conditions based on the method of Rosenthal et al. (1993). The final
reaction volume was 100 µl in 1× buffer (10 mM Tris-HCl, pH 8.3,
50 mM KCl, 1.5 mM MgCl2, 0.005% Tween 20 (v/v), 0.01% (w/v)
gelatine), containing dNTPs (125 µM each), primers (0.1-0.2 µM
each) and 2.5 units of Taq polymerase (AmpliTaq, Perkin Elmer
Cetus), using 0.1-1.0 µg of genomic DNA as template. Thermocycling conditions were: 40 cycles of 1 minute at 94°C, 2 minutes at
55°C and 5 minutes at 72°C each, followed by an extension time of
10 minutes at 72°C.
Three primers specific for the flo gene upstream of the Tam3
insertion site were used:
FloU1 (5′-GCCATCAATTGCATGCATGAAA-3′)
FloU2 (5′-GGTTGTCGGAGGAGCCGGTGC-3′)
FloU3 (5′-GGAAGACGACGAAAATATCGTAAGCG-3′).
Two downstream primers were:
FloD1 (5′-GCAAGCTTGCTCGTACAAATGAAACA-3′)
FloD2 (5′-CAAATCTCCTACAGTGAG-3′)
The primers specific for the left and right end of the transposable
element Tam3 were:
Tam3L (5′-TGCTCGTGTCCATAAATTGTGA-3′)
Tam3R (5′-GAAGACGCAACCATGCCATATG-3′).
The combination of FloU2 and FloD1 used on wild-type Antirrhinum DNA yields a product of about 320 bp; the combination of
FloU1 and FloD2 gives a product of 1.3 kb (Coen et al., 1990). These
PCR products were sequenced using the dsDNA cycle sequencing
system from BRL according to the manufacturer’s instructions, with
FloD2 or FloU3 (for the opposite strand) as sequencing primers. The
above primer combinations (FloU2-FloD1 and FloU1-FloD2) do not
yield any PCR product when used on DNA from flo-613 plants. To
check the presence of Tam3 in the flo gene, the primer combinations
FloU2-Tam3L and FloD1-Tam3R were used, resulting in PCR
products of 1.1 kb and 600 bp, respectively, if the transposon was
present.
In situ hybridization
Tissue preparation and in situ hybridization experiments were carried
out according to Jackson (1991), with modifications as described in
Coen et al. (1990) and Bradley et al. (1993). Digoxigenin-labelled
RNA probes were prepared using the Boehringer Mannheim nucleic
acid labelling kit, following the manufacturer’s instructions. Bluescript KS(+) and SK(+) vectors containing gene fragments of flo, def
or ple (Coen et al., 1990; Sommer et al., 1990; Bradley et al., 1993)
were linearized and used as templates for the labelling reaction to
produce sense and antisense RNA probes.
RESULTS
Expression pattern of flo in the periclinal chimeras
Twelve plants were derived from flowering sectors through
cuttings; three of these plants (plants 1-3) had near wild-type
flowers, one (plant 4) had an intermediate flower phenotype,
and eight (plants 5-12) had flowers of the extreme class
(Carpenter and Coen, 1994). Plant 4 gave rise to progeny that
segregated for wild-type and flo mutant plants in a 3:1 ratio,
whereas plants 1, 2 and 5-11 only gave rise to flo mutant plants.
Plant 3 produced some families that contained only flo mutant
plants and some families that segregated 3:1 for wild-type and
flo mutant plants. Plant 12 gave no viable seed (Carpenter and
Coen, 1994). To determine the relationship between genetic
behaviour, flower phenotype and flo expression pattern, the 12
plants derived from flowering sectors were analysed by in situ
hybridization. Inflorescences of these plants, as well as those
of wild-type and flo-613 mutants, were sectioned and probed
with digoxigenin-labelled flo antisense RNA.
In these sections, bract primordia and their axillary flower
meristems could be seen on either side of the inflorescence
(Fig. 1A). Each section showed a series of developmental
stages, with the youngest near the top of the inflorescence. Not
all of the meristems could be observed in any one section
because of their spiral arrangement. However, by analysis of
serial sections, the position and number of each meristem
Fig. 1. Expression of flo in meristems of wild type and various
chimeras. Staining is dark blue and tissue is counterstained with
calcofluor (pale blue). The top row shows wild type: (A)
inflorescence apex (B) floral meristem at node 10 and (C) floral
meristem at node 14. Comparable meristems are shown below wild
type for an L1 chimera (plant 2, D-F), L2 chimera (plant 4, G-I) and
L3 chimera (plants 8 (J,K) and 9 (L)). In the case of the L2 chimera,
a bract primordium rather than a floral meristem is shown in the
middle panel (H). Abbreviations are: B, bract; F, flower meristem; S,
sepal primordium. Scale bars, 50 µm.
floricaula in single cell layers activates downstream genes
A
B
C
D
E
F
G
H
I
J
K
L
29
30
S. S. Hantke, R. Carpenter and E. S. Coen
relative to the first visible bract primordium (node 0) could be
determined.
At early developmental stages (nodes 0-10), expression of
flo was seen in young bract primordia and their associated
floral meristems. Expression was observed in all three cell
layers of wild type (Fig. 1A,B) but was not detected in flo-613
plants (not shown). Two of the plants with near wild-type phenotypes showed strong expression only in the epidermal layer,
indicating that they were chimeras in which flo activity had
been restored only in L1 (L1 chimeras, plants 1 and 2, Fig.
1D,E). The third near wild-type plant gave variable results:
some inflorescences showed expression in L1 alone whereas
others showed expression in all three layers. This was consistent with the variable results obtained from progeny testing of
this plant (plant 3, Table 1). One possible explanation is that
this plant was originally derived from an L1 chimera and that
further excisions or layer invasions resulted in part of it
expressing flo in all layers. The plant with the intermediate
phenotype showed strongest expression in the subepidermal
layer of cells, indicating that flo activity had been restored in
L2 (L2 chimera, Fig. 1G,H). This was consistent with the
progeny testing on this plant, which showed that a wild-type
allele was present in its germ line (Table 1). Most of the
extreme plants showed relatively little signal in either of the
outer two cell layers but strong expression in the remaining
core of the meristems, corresponding to L3 (plants 5-11, Fig.
1J,K). One extreme plant showed equally strong expression in
both L1 and L3 (plant 12, data not shown). In summary, the
phenotype of the chimeras depended on the layer expressing
flo: L1 chimeras were near wild type, the L2 chimera was intermediate and L3 chimeras were extreme.
By about nodes 13-14, wild-type floral meristems comprised
a central dome with sepal primordia on its periphery (Fig. 1).
In wild type, flo expression was present in all layers of the sepal
primordia and in a small area where petal primordia were initiating (Fig. 1C; Simon et al., 1994). At a comparable morphological stage, the signal in L1 chimeras was in the
epidermal cells of the sepal and petal primordia and in some
sections extended over the central dome (Fig. 1F). The L2
chimera showed flo expression in almost all of the cells in the
sepal, except for the epidermis (Fig. 1I). This is consistent with
previous cell lineage studies showing that most internal cells
of the sepal are clonal descendants of L2 (Satina and Blakeslee,
1941). The signal in L3 chimeras was largely excluded from
sepal primordia and confined to the core cells of the meristem.
In some L3 chimeras, expression extended further towards the
centre of the dome than in wild type (Fig. 1L).
During the next stages of wild-type development (nodes 1520), the expression of flo declined in bracts but was observed
in all floral organs except stamens. After node 20, expression
declined first in sepals and then in petals and carpels. In the
chimeric plants, expression mirrored that in wild type, except
that it was restricted to their respective layers.
Southern blot analysis of DNA from chimeras and
their progeny
The genetic, phenotypic and in situ analysis indicated that
excision of Tam3 gave rise to revertant sectors having different
genetic constitutions in their layers. To test this further, the
genomic DNA content of the chimeras was characterised and
compared with that of their progeny. Total DNA of each
Table 1. Summary of phenotypic, molecular and genetic
behaviour of chimeric plants
Progeny
Flower
phenotype
Plant
number
Near
wild type
1
L1
flo
flo-613
Flo+
2
L1
flo
Flo+
3
4
L1†
L2
flo†
3:1‡
5
6
L3
L3
flo
flo
7
8
9*
L3
L3
L3
flo
flo
flo
10*
11
12
L3
L3
L1+L3
flo
flo
ND
flo-613 and
rearrangement
Imprecise excision†
Flo+ for wt plants
flo-640 for
flo plants
Imprecise excision
flo-613 and
imprecise excision
flo-613
flo-613
flo-613 and
rearrangement
ND
ND
ND
Intermediate
Extreme
Layer of
flo expr. Phenotype
Main
excision
allele in
chimera
Genotype
ND
ND
flo-640
flo-640
flo-670
Flo+
flo-640
ND
flo-669
ND
*Plants 9 and 10 had a particularly extreme phenotype.
†Some apices of plant 3 expressed flo in all cell layers and accordingly
some of the capsules sown segregated for wild type and flo mutant in a 3:1
ratio. In these cases, the alleles in the progeny were Flo+ for wild type and
flo-640 for flo mutant plants.
‡All progeny obtained from plant 4 segregated in a 3:1 ratio for wild type
and flo mutant phenotype.
ND, Not determined, either because no progeny was available at the time,
or because two different excision alleles were present in the original chimera,
or because the sequence of the wild type size PCR product was ambiguous.
chimera should reflect the combined genotype of all layers,
whereas the DNA of its progeny should reflect that of L2. To
distinguish flo-613 from alleles lacking Tam3, Southern blots
of genomic DNA digested with EcoRI were probed with flo
(Fig. 2). This gave a 9.0 kb band for flo-613 and a 5.5 kb band
for wild-type plants that lack the 3.5 kb Tam3 element (Fig.
2A, left two lanes). Most of the chimeras had a 9.0 kb band,
showing that they carried Tam3 in the flo gene. In addition, all
chimeras had a 5.5 kb band of varying intensity, presumably
because some of their cells lacked Tam3 at the flo gene, as a
consequence of excision (Fig. 2A). In several cases, the
intensity of the 5.5 kb band was quite weak, as would be
expected if only one layer was heterozygous for the excision
allele (plants 2,7,8,10,11). The progeny of several L1 or L3
chimeras did not show the excision band, demonstrating that
the excision allele had not been transmitted through the germ
line (plants 1,2,7,8). However, the excision allele was present
in the progeny of both plants that gave germ line revertants
(plants 3,4).
In addition to the flo-613 allele and excision alleles restoring
flo wild-type activity, two further types of allele were observed
in some chimeras and their progeny. In several cases, flo
mutant progeny were observed to have the 5.5 kb excision band
instead of the expected 9.0 kb size (plants 3,4,5). Furthermore,
the intensity of the excision band in the total DNA from several
of the chimeras was much greater than expected if only one
layer had an excision allele (plants 3,4,5,6,9). This was particularly striking for plant 5, in which all layers appeared to have
an excision allele even though it was an L3 chimera. These
floricaula in single cell layers activates downstream genes
31
A
B
results were most likely due to imprecise excisions restoring
the wild-type fragment size but not flo function (see below).
More complex rearrangements were present in the flo gene
of plants 2 and 9. Both of these plants had an additional band
at about 13 kb which was transmitted to their progeny. Many
types of rearrangements involving Tam3 have been described
previously (Martin et al., 1988; Robbins et al., 1989; Bollmann
et al., 1991), but the precise nature of those in plants 2 and 9
have not been determined.
Sequence of the excision alleles in the chimeras
Although there was a correlation between the layer expressing
flo and the phenotype of the flowers, the layer of expression
did not account for all aspects of phenotypic variation. Considerable variation in phenotype was observed between
different plants of the extreme class. Furthermore, plant 12
expressed flo in both L1 and L3, yet it had an extreme
phenotype rather than near wild type. One possible explanation for these discrepancies was that imprecise excision of
Tam3 left footprints that restored flo function to various
degrees. To test this, excision alleles were amplified by PCR
from total DNA of chimeras and their progeny, and the
sequence of the PCR products was determined (Table 1 and
Fig. 2. (A) Southern analysis of chimeras and their
progeny. Genomic DNA was isolated from each
individual chimera (C) and from a pool of 15 progeny
plants (P) of each chimera. Samples of 5 µg DNA were
digested with EcoRI, blotted and probed with flo. The
type of chimera (L1, L2, L3) and plant numbers (1-11)
are given above the lanes. Controls are shown in the left
two lanes: DNA of wild-type plants (wt) gives a 5.5 kb
band and DNA of flo-613 plants (flo-613) gives a 9.0 kb
band. Note that flo-613 also has a weak band at 5.5 kb
because the DNA was derived from plant material grown
in cool outside conditions which favour somatic excision
of Tam3. (B) Map of the flo-613 allele to illustrate the
origin of fragments observed in Fig. 2A. The flo-613
allele carries an insertion of the transposon Tam3 (3.5 kb)
in the second exon of the flo gene. Restriction enzyme
digest with EcoRI results in a 9.0 kb fragment if Tam3 is
present, and in a 5.5 kb fragment if Tam3 is absent. A 1.5
kb fragment from the 3′-end of the gene is detected in
EcoRI digests when blots were probed with the flo cDNA,
irrespective of the presence of Tam3. The thin line
indicates the flo genomic sequence, filled boxes represent
exons, the Tam3 insertion is indicated by a triangle,
EcoRI restriction sites are marked with E and the
transcription start site is indicated by a wavy arrow.
Fig. 3). Precise excisions, giving the wild-type sequence, were
observed in L1 and L2 chimeras and in one L3 chimera. This
confirmed that the phenotype of these plants was a direct consequence of the layer of Flo+ expression. Plant 7 carried an
imprecise excision, flo-670, that restored the original reading
frame but resulted in a conservative amino acid substitution.
Plant 11 carried the flo-669 footprint, which could code for a
protein with a three amino acid insertion relative to wild type.
Plants predicted on the basis of Southern blots to carry additional imprecise excisions (plants 3,4,5,6,9) contained an 8 bp
footprint whose sequence was identical to that of the previously described stable flo-640 allele, which had a frameshift
and encoded a truncated protein. Unfortunately, it was not
possible to determine the sequence of the functional flo alleles
in plants 5, 6 and 9 because of the abundance of the flo-640
sequence that was always present in an excess after PCR.
Expression of organ identity genes in the chimeras
Expression of flo in only one layer seemed to be sufficient to
activate the floral genetic programme, resulting in the
formation of flowers. However, the phenotypes observed
indicated that this activation was less effective than in wild
type. To determine whether this might have been due to altered
32
S. S. Hantke, R. Carpenter and E. S. Coen
Fig. 3. Sequence of flo alleles detected in chimeras compared to Flo+
and flo-613. The target duplication caused by insertion of Tam3
(triangle) in flo-613 is indicated by capital letters and by an arrow
above the sequence. Alterations relative to the Flo+ sequence are
highlighted in bold. The predicted amino acid sequences are shown
under each allele in the one-letter code.
expression of later acting genes, we analysed the expression of
the organ identity genes, deficiens (def) and plena (ple)
(Sommer et al., 1990; Bradley et al., 1993) in the chimeras. As
controls, adjacent sections were also probed with flo to confirm
which layer expressed flo (Fig. 4A,D,G,J). The results were
compared with those obtained with wild type and flo mutants.
In wild type, expression of def was first detected at about
node 11, and by node 13 def was strongly expressed in all cell
layers of the central dome, resembling two inverted peaks in
median sections (Fig. 4C). After this stage, the def gene
continued to be expressed strongly in the developing petal and
stamen primordia (Fig. 4F,L). In flo-613 and flo-640 mutants,
no expression of def was observed (data not shown), showing
that flo activity is normally required to switch on def. In the
chimeras, def was expressed in all three cell layers but the
timing and distribution was different from wild type. Detailed
analysis was carried out on L1 and L3 chimeras only, since
insufficient amounts of L2 material were available.
In L1 chimeras, expression of def was first observed at about
nodes 14-15 (Fig. 4B) corresponding to a delay of about three
nodes compared to wild type. By node 16, def was strongly
expressed in two inverted peaks, similar to the pattern seen in
node 13 of wild type (Fig. 4E). However, the def domain
appeared to be more restricted than wild type and did not extend
all the way to the centre or outer edges of the dome (compare
Fig. 4C with E). A significant delay in morphological development was observed in L1 chimeras by the analysis of serial
sections as well as by scanning electron microscopy (Carpenter
and Coen, 1994). Due to this delay, the overall appearance of
node 16 meristems of the L1 chimera was similar to node 13
of wild type: in both cases, sepal primordia surrounded a central
dome (compare Fig. 4C with E). However, node 16 meristems
of the L1 chimeras were larger than node 13 of wild type. The
reduced domain of def expression may therefore reflect the
increased meristem size at the time of def activation. In contrast
to node 16 of L1 chimeras, node 15 meristems of wild type
showed distinct petal and stamen primordia expressing def and
a flattened central region void of def expression (Fig. 4F).
During later stages, from about node 20 onwards, morphology
of meristems and def expression in L1 chimeras was similar to
wild type.
In L3 chimeras, def expression was first detected in nodes
13-14, corresponding to a delay of two to three nodes as
compared to wild type, correlating with their retarded development observed by scanning electron microscopy (Carpenter
and Coen, 1994). The def domain in nodes 14-15 resembled
that of wild-type node 13 meristems, forming two inverted
peaks with no gap in the centre (Fig. 4H). However, the boundaries of the def domain did not extend to the outer edges of the
central dome. In some of L3 chimeras the delay in def
expression was greater than two to three nodes and could be
as much as eight nodes in plants with the most extreme phenotypes (plants 9 and 10).
At later stages of L3 chimera development, def expression
remained low in some regions of petal primordia, particularly
in the outer cells. In most cases, def expression eventually
attained the wild-type pattern by nodes 16-22. However, in
plants with particularly extreme phenotypes, abnormally
restricted streaks of def expression could be observed along the
axis of petals as late as nodes 23 or 24 (Fig. 4K). In stamens,
def seemed to be expressed normally.
Expression of ple in wild type was first detected at about
node 12 and was observed in all cell layers of the central dome
of the floral meristem by nodes 13-14. After this stage, ple
continued to be strongly expressed in the developing primordia
of whorls 3 and 4. In flo-613 and flo-640 mutants, no
expression of ple was observed (data not shown), demonstrating that flo activity is normally required to switch on ple. In
chimeras, expression of ple was observed in all cell layers but
was delayed by about 2-3 nodes. Once activated, ple
expression had a similar pattern to wild type (Fig. 4I).
DISCUSSION
We show by in situ hybridization, DNA and sequence analysis
Fig. 4. Expression of flo, def and ple in periclinal chimeras.
Consecutive sections were probed with digoxigenin-labelled flo, def or
ple antisense RNA and viewed as in Fig. 1. An L1 chimera (plant 1) is
shown probed with flo and def at node 15 (A and B resepectively) and
node 16 (D and E respectively). For comparison, expression of def in a
wild-type node 13 (C) and node 15 (F) are shown. Note the differences
in def expression pattern and signal strength between B and C even
though the morphologies appear comparable. In comparing E and F,
note the more advanced floral organ primordia in the wild type and the
difference in the def expression domain. An L3 chimera node 14 floral
meristem (plant 5) is shown probed with flo (G), def (H) and ple (I).
The def domain is similar to that in node 13 of wild type (compare H
and C) but does not extend as far towards the sepal primordia. A node
23 floral meristem of an L3 chimera (plant 10) showing expression of
flo (J) and def (K) compared to a node 21 of wild type probed with def
(L). Expression of flo can hardly be detected in the chimera at this
stage whereas that of def is reduced to narrow streaks in petals, but is
apparently normal in stamens (compare K and L). Abbreviations:
c, carpel; p, petal; s, sepal; st, stamen. Scale bars (shown once per row)
is 100 µm.
floricaula in single cell layers activates downstream genes
that the flowering sectors on flo-613 mutant plants are periclinal chimeras for flo caused by excision of Tam3. The excision
events occur early in meristem development, restoring the
33
expression of flo in only one cell layer. Among the 12 plants
derived from flowering sectors (Carpenter and Coen, 1994)
three main types of chimera were identified: L1 chimeras
A
B
C
D
E
F
G
H
I
J
K
L
34
S. S. Hantke, R. Carpenter and E. S. Coen
express flo in the epidermal layer of the meristem, the L2
chimera expresses flo in the subepidermal layer and shows
germ line transmission of the revertant allele, and L3 chimeras
express flo in meristem core. The types of chimera correlate
with the flower phenotypes: L1 chimeras are near wild type,
the L2 chimera are intermediate, and L3 chimeras have an
extreme phenotype. Tam3 excision events can be detected in
the chimeras both by Southern blots and DNA sequencing,
confirming that they arose by transposition.
These results demonstrate that flo expression in single cell
layers can promote flower development in a non-autonomous
manner, coordinating cell division and differentiation in all
layers.
However, some aspects of flo function are hampered when
it is restricted to one layer, evidenced by two observations.
Firstly, the flower phenotypes of the chimeras are not normal:
L1 chimeras give near wild-type flowers, the L2 chimeras are
intermediate and L3 chimeras give an extreme phenotype.
Secondly, the chimeras with near wild-type or intermediate
phenotypes all carry Flo+ alleles caused by precise excisions,
even though such events are likely to be rare (Coen et al., 1989,
1990). This suggests that when flo function is hampered by
restriction to one layer, the requirements to produce nearly
normal flowers are so stringent that precise excisions are
selected for. This is supported by the observation of imprecise
excisions in some of the chimeras with extreme phenotypes.
Interactions of flo with downstream organ identity
genes
At least two organ identity genes, def and ple, are activated in
all three layers of flo chimeras, even though they are not
activated in stable flo mutants. This indicates that the nonautonomy of flo is effective very early in floral development
and that activation of organ identity genes by flo involves cell
communication between layers. Such communication may also
be involved in coordinating the expression pattern of organ
identity genes between the layers.
Although extending to all layers, the def expression domain
is reduced, and the activation of both def and ple appears to be
delayed in the chimeras. The effects on def are even greater in
L3 chimeras with particularly extreme phenotypes. These
effects on def and ple gene expression may partly explain the
phenotype of the chimeric flowers. For example, def mutations
result in sepals replacing petals in whorl 2 and carpels
replacing stamens in whorl 3. The altered pattern of def
activity, particularly in whorl 2, may therefore account for the
petals in some chimeras being split, small, and having streaks
of green, sepaloid tissue. The expression domain of ple, a gene
controlling sex organ identity, is not markedly affected, consistent with the ability of the chimeras to produce fully functional sex organs. These results suggest that expression of def,
particularly in whorl 2, is relatively sensitive to changes in flo
activity. This may reflect the properties of fimbriata, a gene
known to mediate between flo and def (Simon et al., 1994). A
close dependence of def on flo is also suggested from the
analysis of counterpart genes in Arabidopsis, which shows that
apetala3 expression is reduced in leafy mutants (Weigel and
Meyerowitz, 1993). However, it is unlikely that all aspects of
chimera phenotype can be accounted for by altered def and ple
activity. The altered morphology of first whorl organs
(Carpenter and Coen, 1994), for example, suggests a role of flo
in the activation of genes involved in specifying sepal identity.
Autoregulation of flo
Analysis of plants carrying the flo-640 allele indicates that flo
increases its own expression (unpublished data). The flo-640
allele carries a frameshift caused by imprecise excision of
Tam3 and therefore has the potential to be transcribed
normally. Nevertheless, when compared to wild type, plants
homozygous for the stable flo-640 allele express flo at low
levels, providing evidence for flo autoregulation. This raises
the question of whether restoring flo activity in one layer can
increase flo expression in other layers. This could be tested
because several of the chimeras contain alleles with the same
sequence as flo-640 in layers other than the one with restored
flo function. For example, two of L3 chimeras contain substantial amounts of the flo-640 sequence, and this appears to
be transmitted to their progeny, showing that L2 and possibly
other layers carry this sequence. However, flo transcripts are
mainly observed in L3 of both of these chimeras, reflecting the
location of the functional allele. This suggests that flo activity
in L3 cannot increase expression of flo in other layers,
otherwise strong expression would also be seen in the L1 and
L2 of these chimeras. These results indicate that flo autoregulation is largely cell-autonomous with respect to the layers.
Another aspect of flo autoregulation is suggested by the
more extensive flo expression pattern observed in some of the
chimeras at early stages of development. For example, flo
expression in wild-type meristems at about node 13 is not
observed in the main part of the central dome, destined to form
whorls 3 and 4. However, at a comparable stage in L1 and L3
chimeras, flo expression sometimes extends further into the
central dome than in wild type. One explanation for this is that
flo normally activates organ identity genes, which are responsible for inhibiting flo activity in the central dome, at an early
stage (Coen et al., 1990). In the chimeras, there may be a delay
in the activation of such genes and hence ectopic expression
of flo. This is supported by the observed delay in expression
of organ identity genes such as def and ple in the chimeras.
The above results illustrate how the molecular analysis of
chimeras can provide important information about the role of
cell communication within and between meristematic layers
and how flo interacts with itself and other genes to control
flower development. It should be possible to use the same
approach to analyse chimeras for other developmental genes to
give a much more detailed picture of the overall control of
floral morphogenesis and the cell interactions involved.
We are grateful to H. Sommer for kindly providing the def clone
used for production of RNA probes. For critical reading of the manuscript and constructive comments, we thank Desmond Bradley, Pilar
Cubas, Sandra Doyle, David Hopwood, Gwyneth Ingram, Paula
McSteen, Jackie Nugent, Sylvie Pouteau, Elisabeth Schultz and
Yongbiao Xue. We are also grateful to the AFRC stem cell initiative,
Boehringer Ingelheim Fonds and HFSPO for financial support.
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