Abteilung für Molekulare Pflanzengenetik, Max-Planck-Institut für Züchtungsforschung, 50829 Köln, Germany
Author for correspondence (e-mail:
sommer{at}mpiz-koeln.mpg.de)
Accepted 29 September 2004
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SUMMARY |
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Key words: MADS-box protein, Prophyll, Floral meristem identity, Floral architecture, Antirrhinum majus
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Introduction |
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Vegetative and reproductive organs are arranged in a species-specific
phyllotaxis (Reinhardt and Kuhlemeier,
2002). The switch from vegetative to reproductive growth is
accompanied by a change of phyllotaxis in Antirrhinum majus
(Carpenter et al., 1995
).
During the vegetative phase, the plants display decussate phyllotaxis, where
the two leaves formed per node are positioned at opposite sides of the stem.
After the transition to the reproductive phase, Antirrhinum plants
switch to a spiral phyllotaxis, producing small leaf-like organs (bracts) at
each node along the main (inflorescence) stem. Flowers arise in the axils of
bracts and consist of four types of organs arranged in a whorled phyllotaxis.
Five sepals in the calyx constitute the outer (first) whorl, followed by five
petals (second whorl), four stamens and a stamenoid (third whorl), and two
fused carpels in the inner (fourth) whorl. Mutations in two
Antirrhinum genes, FLORICAULA (FLO) and
SQUAMOSA (SQUA), transform flowers into indeterminate
axillary inflorescences with bracts arranged in spiral phyllotaxis
(Coen et al., 1990
;
Huijser et al., 1992
). The
phenotype of flo and squa mutants indicates that both genes
play a crucial role in the specification of the floral meristem. The
transcript level of SQUA and FLO in flo and
squa mutants, respectively, is unchanged, but after independent
transcriptional induction, the SQUA and FLO functions converge in the control
of flower development. This is revealed by the enhanced squa or
flo mutant phenotypes when the respective FLO or
SQUA functions are reduced
(Carpenter et al., 1995
). In
this report, we provide genetic evidence that INCOMPOSITA
(INCO) is an additional factor required, in cooperation with
FLO and SQUA, for the control of floral meristem
identity.
INCO, like SQUA, is a MADS-box transcription factor
(Schwarz-Sommer et al., 1990).
MADS-box genes constitute a large family, which, throughout plant evolution,
have been recruited as transcriptional regulators controlling the development
of various plant structures and organs (Ng
and Yanofsky, 2001
). inco flowers display two extra
organs, named prophylls or bracteoles
(Bell, 1991
;
Weberling, 1989
), which
develop very close to the lateral sepals. We propose that INCO
represses prophyll development in Antirrhinum, which is a novel
function for a MADS-box transcription factor, and show that absence of this
control results in impaired floral architecture.
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Materials and methods |
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Col0 Arabidopsis thaliana plants were transformed according to a
dipping protocol (Clough and Bent,
1998). 35S::INCO was constructed by inserting the full-size cDNA
into the BamHI site of the pPCV072 vector
(Koncz et al., 1990
), and for
35S::SVP the XbaI site of pBAR-35S [modified from Becker et al.
(Becker et al., 1992
)] was
used. The T2 progeny of several transgenic lines was grown in climate chambers
at 20°C and 16 hours of light.
Microscopy
Scanning electron microscopy (SEM) was carried out with replicas of flowers
and developing inflorescences as previously described
(Green and Linstead,
1990).
The vascular skeleton was viewed under bright field after processing the
tissues according to Candela et al.
(Candela et al., 1999).
In situ hybridisation and northern blotting
In situ hybridisation with digoxigenin-labelled antisense RNA was performed
as previously described (Bradley et al.,
1993; Davies et al.,
1996
; Huijser et al.,
1992
). The INCO probe did not contain the MADS-box.
For northern blot analyses, 1 µg of mRNA was loaded per lane,
transferred to nylon membranes and processed as previously reported
(Sommer et al., 1990).
DNA preparation and PCR screening
Leaf samples were ground in liquid nitrogen and suspended in extraction
buffer (250 mM TrisHCl, pH 7.5; 1% SDS; 25 mM EDTA, 250 mM NaCl). After
phenol/chloroform extraction, the DNA was precipitated and the pellet was
resuspended in TE buffer containing 5 µg/ml RNAseA. The screening procedure
followed the protocol described by Keck et al.
(Keck et al., 2003). Detailed
information on PCR primers and PCR conditions for these and all other
experiments performed in this report are available upon request.
Yeast methods
For yeast two-hybrid experiments the INCO or SQUA coding sequences were
inserted into the EcoRI/SalI site of the pGAD424 and pGBT9
(Clontech) vectors. INCOC (amino acids 1 to 206) was constructed by PCR
amplification of the respective region of the INCO cDNA. This C-terminal
deletion eliminates the transcriptional activator domain and prevents
auto-activation in yeast. Ternary complex formation was tested with
INCO
MIK1/2 (amino acids 104 to 229 in the pGAD424 vector) using the
full-size PLE cDNA (inserted into pGBT9) and the full-size DEFH200 cDNA
(cloned into the EcoRI/SalI site of the pTFT1 vector
(Egea-Cortines et al., 1999
).
Yeast libraries, screening protocols and all other constructs are described
elsewhere (Davies et al., 1996
;
Egea-Cortines et al.,
1999
).
Semi-quantitative assays for comparing the strength of INCO
homodimerisation and heterodimerisation with several partners were performed
by liquid lacZ assays (Kippert,
1995) using the SFY527 yeast strain. Activity in Miller units was
calculated according the formula (1000
A420
Vr)/(t
Vc
A600), where
Vr=final reaction volume in ml; Vr=volume of culture
assayed in ml; t=time in minutes. Average and standard deviation of four
independent assays are shown in Table
1.
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Results |
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INCO is expressed during early stages of organ initiation
Northern blot and RT-PCR analyses indicate that INCO mRNA is
present in all organs during the vegetative and reproductive phases (not
shown). INCO mRNA is detectable in situ in two opposite domains of
the SAM (Fig. 3A),
corresponding to the position of incipient leaf primordia [P0
(Waites et al., 1998)].
Similarly, INCO transcript is present in initiating bract primordia
in the IM (Fig. 3B).
Meristematic cells within the apical dome of the SAM and IM, however, do not
express INCO. INCO is transcribed in emerging axillary meristems
during the vegetative phase and in floral meristems until early stage 3
(Fig. 3C-E). During stage 3,
INCO mRNA disappears from the emerging sepal primordia and becomes
more restricted to deeper layers of the floral meristem. Later, INCO
expression is only detectable in developing and mature anthers as revealed by
northern blot analysis and in situ hybridisation experiments (not shown).
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INCO represses prophyll development
The most conspicuous feature of inco mutants are two narrow, or
filamentous structures beneath the two lateral sepals, outside the calyx,
which are absent in the wild-type flower
(Fig. 4)
(Wilkinson et al., 2000).
Occasionally, these organs reach the size of sepals, but in contrast to true
sepals, and similar to bracts (Keck et
al., 2003
), they develop a glandular structure at their tip
(Fig. 4D,E). In contrast to
leaves and bracts with branched venation, however, the venation of prophylls
resembles the parallel pattern observed in sepals
(Fig. 4E,F). Mature flowers
frequently display twisted or distorted petals, petaloid lateral sepals and
petals that are partly fused to sepals
(Fig. 4B,C). The lateral sepals
are often smaller than in the wild type and are sometimes reduced to tiny
narrow organs. The inco phenotype is variable, displaying nearly wild
type to severely distorted petals and extra organs that are free-standing or
fused to the adjacent lateral sepals. The phenotypes of different
inco mutants are very similar, although in inco-4 flowers
the additional two organs are sometimes positioned lower on the pedicel or
very close to the subtending bract.
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Severe flo-640 mutants (Fig.
6C) display bracts arranged in a spiral phyllotaxis that carry,
instead of flowers, axillary inflorescences composed of bracts in their axils
(Coen et al., 1990). This
severe phenotype is not affected when combined with inco (not shown).
However, in combination with the weak flo-662 allele, which displays
wild-type-like inflorescences with flowers
(McSteen et al., 1998
),
inco flo-662 double mutants exhibit inflorescences
(Fig. 6D,E). Expression of
FLO is not altered in the inco mutant
(Fig. 3H), indicating that the
enhanced flo mutant phenotype is not due to impaired transcriptional
regulation of flo-662 in the double mutant. The synergistic effect of
mutations in inco and flo thus suggests that the
INCO and FLO functions converge in establishing the floral
meristem. It is possible, that the role of INCO in preventing prophyll
development is related at least in part to its function in the control of
floral meristem identity. If so, then prolonged delay in floral determination
should result in more leaf-like organs before sepal initiation. Frequent
development of an additional filamentous leaf-like organ (`third prophyll') in
the inco flo-662 double mutant (not shown) supports this
assumption.
squa mutants are affected in the transition from inflorescence to
floral meristem, similar to flo mutants, but squa deletion
mutants occasionally produce flowers, which are frequently misshapen and are
subtended by prophyll-like organs (Huijser
et al., 1992). Intriguingly, inco squa plants produced
more flowers and flowered earlier than squa
(Fig. 6F,G), and squa
plants heterozygous for INCO had a phenotype intermediate between
squa and the inco squa double mutant, indicating a dose
effect of INCO.
The partial epistasis of inco to squa might suggest that INCO in the squa mutant background prevents flower formation and that SQUA counteracts this negative influence in the wild type. Enhanced flowering tendency of inco squa double mutants, if interpreted as indication for improved floral determination (see above), should be accompanied by improved flower morphology. Yet, the morphology of inco squa double mutant flowers (Fig. 6H) remains similar to squa flowers, and they even contain a `third prophyll' sometimes. This suggests that promotion of flowering, floral determination and prophyll initiation are not fully linked.
In the squa mutant, INCO transcript was not detectable in
P0 bract primordia (Fig. 3G),
in accordance with the presence of prophylls on the long pedicel of
occasionally forming squa flowers
(Huijser et al., 1992).
SQUA is thus an activator of INCO expression. The level of
INCO expression was slightly reduced in secondary inflorescence
meristems (Fig. 3G), suggesting
that SQUA is not absolutely necessary to establish and maintain
INCO transcription in reproductive axillary meristems.
In summary, the functional relations of INCO, FLO and SQUA in the control of floral meristem identity, promotion of flowering and prophyll formation are complex, and the role of INCO in these processes cannot be simply reduced to the control of floral meristem identity.
Protein interactions
Given the influence of INCO on floral meristem identity, we asked
whether INCO interacts with MADS-box transcription factors involved in the
same process. Yeast two-hybrid screens showed that INCO heterodimerises with
several other MADS-box proteins such as SQUA and the so-called identity
mediating (Im) proteins DEFH200, DEFH84 and DEFH72
(Table 1), the orthologues of
the Arabidopsis AP1 and SEPALLATA (SEP) proteins, respectively
(Egea Gutierrez-Cortines and Davies,
2000). Interestingly, according to semi-quantitative assays,
heterodimer formation between INCO and SQUA, as well as with several other
MADS-box proteins, is favoured compared with INCO homodimer formation
(Table 1). It is likely
therefore, that INCO homodimerisation is prevented in vivo by interactions
with other proteins. As expression of all potential INCO heterodimerisation
partners tested is controlled by SQUA
(Davies et al., 1996
), INCO
homodimerisation appears to be favoured in the squa mutant
background.
SVP is the closest Arabidopsis relative of the INCO protein (Fig. 2) and, similar to INCO, SVP interacts with AP1, SEPALLATA1 (SEP1) and SEP2, as well as with the respective Antirrhinum proteins (Table 1). The similarity in protein interactions is in line with the observed common features of Arabidopsis plants overexpressing SVP or INCO (see below). In contrast to INCO, however, SVP cannot homodimerise and cannot activate transcription in yeast on its own. In fact, the knockout phenotypes of inco or svp mutants differ, in part perhaps as a consequence of these differences.
INCO interacts with the floral organ identity MADS-box protein PLENA (PLE), and we also observed higher order complexes (e.g. ternary) between INCO, PLE and DEFH200 (Table 1). This could suggest that INCO is involved, together with PLE, in a developmental control function in stamens, in agreement with the expression of INCO mRNA in mature anthers. inco mutant stamens are not visibly affected in their development under our growth conditions, suggesting that other factors can mask here the role of INCO.
Ectopic expression of INCO and SVP in Arabidopsis represses flowering
The observation that heterodimer formation is favoured over INCO
homodimerisation prompted us to determine whether an excess of the
INCO gene product resulting in over-production of INCO in the plant
has some developmental consequences. Arabidopsis plants expressing
the 35S::INCO transgene were generated to address this issue.
35S::INCO transgenic plants showed dramatic delay in the floral transition
compared with wild-type plants (Fig.
7A). Their flowers displayed leaf-like features, such as branched
trichomes on sepals, petals and carpels
(Fig. 7B-D). Early arising
flowers were more severely affected than later ones, and initiated
inflorescences within the gynoecium (not shown). All changes observed point to
incomplete floral transition in the presence of the 35S::INCO transgene. This
is in line with the observed enhancement, compared with squa, of
flowering in inco squa double mutants and supports the idea that, in
the squa mutant, flowering is prevented by an excess of INCO, e.g. by
formation of the INCO homodimer. In fact, Arabidopsis svp mutants
flower earlier than wild type, suggesting that SVP prevents floral transition
(Hartmann et al., 2000). In
agreement with this function, and also with the yeast experiments, the
behaviour of 35S::SVP and 35S::INCO in transgenic plants is similar
(Fig. 7A,D), suggesting some
common molecular and functional properties of the proteins. However, the
svp and inco knockout phenotypes differ, indicating that
this common potential is exploited in different ways in Arabidopsis
and Antirrhinum. Thus, as previously noticed, the bona fide function
of MADS-box proteins cannot be fully deduced from overexpression in transgenic
plants in the absence of observations with loss-of-function mutants
(Davies et al., 1999
).
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Discussion |
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The tomato MADS-box gene JOINTLESS is expressed in all tissues
tested by northern hybridisation, but has a defined role in the establishment
of the abscission zone on pedicels only
(Mao et al., 2000).
SVP, a negative regulator of flowering time in
Arabidopsis, is expressed in reproductive meristems in a pattern
similar to INCO (Hartmann et al.,
2000). Interestingly, overexpression of SVP or
INCO in Arabidopsis prolongs the vegetative phase and
prevents flowering suggesting potential functional similarities.
INCO, like SVP, could either directly repress the vegetative
to floral transition in Arabidopsis, or could indirectly interfere
with the function of proteins controlling flowering. As several regulators of
Arabidopsis flowering time are MADS-box proteins (reviewed by
Becker and Theißen,
2003
), reinforced interactions in the presence of excess of SVP or
INCO might result in their depletion and inactivation. In spite of the
potential to interfere negatively with flowering in Arabidopsis and
in apparent contrast to svp mutants, inco mutants have no
obvious flowering time defects. In fact, as discussed below, the negative role
of INCO is masked by competitive interaction with other proteins, such as
SQUA, in the wild-type plant.
AGL24 and SVP are close Arabidopsis relatives within the StMADS11
group (Fig. 2B). The
AGL24 gene is abundantly expressed in Arabidopsis in all
meristems, except for the floral, where its expression is limited to a single
cell layer (Yu et al., 2004).
When overexpressed, AGL24 promotes flowering
(Michaels et al., 2003
), in
apparent contrast to INCO and SVP, which repress flowering
(see above). Furthermore, in the wild type, AGL24 negatively controls
floral meristem identity as demonstrated by the rescued, wild-type like
phenotypes of double mutants of agl24 with lfy
(Yu et al., 2004
). By
contrast, defects in FLO (the orthologue of LFY) in
flo mutants are enhanced by inco, suggesting a positive role
of INCO in the control of Antirrhinum floral meristem
identity, as discussed below. Thus, the role of INCO in floral
meristem identity control is distinctively different from that of either
SVP or AGL24.
Last but not least, INCO is a crucial control gene for repression of prophyll development, a function that has not been reported yet for MADS-box genes. Thus, the StMADS11 clade unites members with a variety of different functions: some are unique to individual members, some are shared among them and some others have opposite functions.
Prophylls: some plants have them and some do not
The term prophyll is applied to the leaf or leaves at the first (proximal)
node on the shoot, distinguishable in shape and arrangement from other leaf
organs. Bracteoles, which are small leaf-like organs developing between a
subtending bract and the calyx of the flower, are therefore prophylls
(Bell, 1991). Prophylls
possibly protected floral buds and reproductive organs in ancestors of modern
angiosperms, and their function was taken over by the calyx during evolution.
The evolutionary ancient origin of prophylls is supported by their presence in
the `living fossil' Amborella, where female and male flowers contain
two prophylls placed close to, or within, the flower
(Endress and Igersheim, 2000
).
Similar to inco mutant flowers Amborella prophylls can fuse
with the nearest sepals.
Prophylls became integrated into the calyx of some species during evolution
and in some others prophylls were `lost'
(Weberling, 1989). Indicative
for the former case are flowers where lateral sepals (the genuine position of
the prophylls) initiate first, while evolutionary loss (degeneration by
suppression) of prophylls is suggested by the abaxial-dorsal-abaxial sepal
initiation pattern, before lateral sepals appear. In Antirrhinum, the
order of sepal initiation in the wild type indicates that prophylls were not
integrated into the calyx and the development of prophylls in the
inco mutant suggests that INCO was recruited during
evolution to prevent their development. This interpretation correlates well
with the lateral position of inco prophylls beneath the calyx, the
emergence of their primordia before those of ventral and dorsal sepals, and
the maintenance of the principal order of floral organ initiation in the
inco mutant. Alternatively, it is possible that the inco
mutation affects the timing of lateral sepal initiation and causes a
heterochronic homeotic defect. In this scenario, the lateral sepals initiate
first, at a time when floral identity is not fully established, and hence they
acquire an intermediate bract/sepal fate. The pair of lateral organs that
develop subsequent to the ventral and dorsal sepals correspond then to extra
sepals, which, in the mature calyx, occupy the position of lateral sepals. The
developmental role of inco thus would be to prevent premature lateral
sepal initiation. However, it is difficult to relate displacement of the two
first initiated (transformed) sepals outside of the calyx and initiation of
two extra organs to a homeotic defect alone. Therefore we favour the `prophyll
hypothesis', where the extra inco organs are prophylls rather than
homeotically transformed lateral sepals.
Several species of the Scrophulariaceae lack prophylls, like
Antirrhinum, but their presence is common as well
(Heywood, 1998), for example,
in two of the English Verbascum species
(Stace, 1997
) and in the
Chinese Mimulus bracteosus. It will be interesting to elucidate
whether presence and absence of prophylls in a species is associated with
changes at an INCO-like locus.
Prophyll development and floral architecture
Various theories assume that organ initiation is regulated by the geometry
of the apex and by mechanical forces (tension and compression) that act on
meristem surfaces (Reinhardt and
Kuhlemeier, 2002). According to the theory of the `first available
space', based on microsurgical manipulations, the timing and positioning of
organ initiation is regulated by the availability of the minimal free area on
the meristem surface, at a minimal distance from the summit and from
pre-existing primordia (Snow and Snow,
1931
; Snow and Snow,
1933
). In other interpretations, space itself is not decisive; a
new primordium emerges at the position of weakest inhibition where the most
recently formed primordium is the strongest source of inhibition
(Tooke and Battey, 2003
;
Wardlaw, 1949
). Indeed, in
inco mutants, development of lateral sepal primordia is significantly
delayed, owing to the aberrant initiation of prophyll primordia, but the
temporal order and the principal site of their initiation are not affected.
Initiation of prophylls thus does not seem to interfere with auxin
redistribution, shown to be a decisive factor in the maintenance of
phyllotaxis (Reinhardt et al.,
2003
).
However, the presence of prophylls has severe consequences for the overall
architecture of the flower, in that lateral sepal primordia are forced towards
the developing petal primordia in the second whorl and, perhaps owing to
consumption of cells by the prophylls, lateral sepals are frequently smaller
than the corresponding wild-type sepals. Chimeric sepaloid-petaloid organs
develop frequently, or, if contact is established to the petal primordia,
sepals and petals can fuse. Such anomalies were also observed in sunflower,
where applying mechanical stress during development resulted in large bracts
instead of the dyad (bract/floret) structure
(Hernandez and Green, 1993).
The mechanical nature of these alterations in the inco mutant is
corroborated by the lower frequency of size reduction of organs and of fusions
between first and second whorl organs in inco def double mutants,
where the size of second whorl primordia is reduced, owing to their homeotic
transformation to sepaloid organs. Thus, repression of prophyll initiation by
INCO is a prerequisite for establishment of the wild-type floral
architecture.
INCO is a novel component of Antirrhinum floral meristem identity control
The phenotype of inco with the lack of repression of prophyll
development and the disordered development of floral organs in inco
mutants resembles the phenotype of rarely forming squa flowers. In
fact, we found that during the time of bract initiation, SQUA is a
direct or indirect activator of INCO expression
(Fig. 8, left).
|
Somewhat surprisingly, INCO is also a positive factor in the
control of floral meristem identity. This is uncovered in inco
flo-662 double mutants, where inco enhances the otherwise not
manifested meristem identity defect in the weak flo-662 mutant. In
this respect, the influence of INCO is comparable with the
enhancement of the flo-662 mutant phenotype in the background of
squa [see Introduction and Carpenter et al.
(Carpenter et al., 1995)].
Thus, in the presence of SQUA, INCO acts together with FLO
to promote flower development.
Given that wild-type plants flower in spite of INCO expression and hence potential repression of flowering, we have to explain how INCO can become a positive factor in flowering. One appealing assumption is that the INCO/SQUA heterodimer (and/or heterodimerisation with proteins whose expression is controlled by SQUA) performs the promoting function, and that in the presence of SQUA heterodimerisation is favoured compared with INCO homodimerisation (Fig. 8, green lines). This would deteriorate the repressive function of the INCO homodimer in the wild type, which is in favour of promotion of flowering. In fact, at least in yeast, the SQUA/INCO interaction (and the interaction of INCO with several other SQUA-controlled proteins) is stronger than INCO homodimerisation. In addition, impaired floral determination of transgenic Arabidopsis plants expressing excess of INCO shows that disturbing the balance in favour of INCO, and hence facilitating homodimer formation, enhances the negative effect of INCO. The overlap of the SQUA and INCO expression patterns in initiating floral primordia is in agreement with the potential for protein-protein interaction between the INCO and SQUA proteins in planta.
Intriguingly, in the presence of sufficient SQUA and FLO function in the wild type, the role of INCO in the control of floral meristem identity appears superfluous, and the lack of INCO in the inco mutant manifests itself in prophyll development only. Possibly, suppression of prophyll development by INCO is a relatively novel function acquired by a MADS-box protein with the potential to interfere with the floral transition. The complex relations to SQUA and FLO were perhaps established to prevent this interference.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: CSBT, Istituto Agrario San Michele all'Adige, 38010 San
Michele all'Adige (TN), Italy
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