1 Abteilung für Molekulare Pflanzengenetik, Max-Planck-Institut für
Züchtungsforschung, 50829 Köln, Germany
2 School of Biological Sciences, Monash University, Clayton, VIC 3800,
Australia
3 Institut für Biologie II, Zellbiologie, Universität Freiburg, 79104
Freiburg, Germany
Author for correspondence (e-mail:
schwarzs{at}mpiz-koeln.mpg.de)
Accepted 1 April 2004
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SUMMARY |
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Key words: GRO/TUP1, Co-repressor, Floral organ identity, Leaf development, Auxin
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Introduction |
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The Antirrhinum mutants stylosa (sty),
fistulata (fis), choripetala (cho) and
despenteado (des) display partial loss of control over the
establishment or maintenance of the outer expression domain of the B- and
C-functions in their flowers (McSteen et
al., 1998; Motte et al.,
1998
; Wilkinson et al.,
2000
). This is revealed by petaloid sepals in the first floral
whorl and/or by stamenoid features in the second whorl as a result of ectopic
expression of class B and class C genes. Interestingly, ectopic expansion of
the B and C functions often occurs concomitantly suggesting that the
regulation of their expression may involve common factors. In addition, the
mutants display other abnormal features such as narrow vegetative and floral
organs (in cho and des), or fasciation and aberrant carpels
(in sty). The homeotic defects in the single mutants are not
striking, although they can become more pronounced depending on the genetic
background. Double mutant combinations, however, display severely enhanced
homeotic phenotypes in all genetic backgrounds. This suggests that STY, FIS,
CHO, DES and some additional factors function together, perhaps as components
of a larger protein complex, or in independent pathways that converge to
control the outer limits of the B and C domains.
In this report we show that STYLOSA (STY) is the
orthologue of LEUNIG (LUG), an Arabidopsis gene
that represses the C-function gene AGAMOUS in the two outer whorls
(Conner and Liu, 2000;
Liu and Meyerowitz, 1995
). The
STY and LUG proteins are structurally related to GRO/TUP1-like corepressors
found in all metazoans and yeasts (Conner
and Liu, 2000
). GRO/TUP1 interact with diverse DNA-binding
partners and are involved in regulation of a broad range of developmental
processes (reviewed by Fisher and Caudy,
1998
). One such partner in Drosophila, mammals and yeast
is represented by a heterogeneous group of proteins that contain a DNA-binding
HMG box (Brantjes et al., 2001
;
Cavallo et al., 1998
;
Deckert et al., 1995
). This
association appears to be important for the formation of larger nucleoprotein
complexes, termed `repressosomes', where HMG-box proteins represent
architectural factors (Courey and Jia,
2001
). We found that STY interacts in yeast with GRAMINIFOLIA
(GRAM), a member of the plant-specific YABBY protein family
(Golz et al., 2004
). YABBY
proteins have a highly conserved N-terminal zinc-finger domain and a truncated
HMG domain (the YABBY domain), whereas the internal region between these
domains and the C terminus are variable
(Bowman and Smyth, 1999
;
Sawa et al., 1999b
). In vitro
DNA-binding studies with the YABBY protein FILAMENTOUS FLOWER (FIL) showed
that the HMG box is essential for protein-DNA interaction and the zinc-finger
domain stabilises the protein structure
(Kanaya et al., 2002
).
GRAM together with other YABBY proteins such as PROLONGATA (PROL) is involved in the control of leaf polarity and growth. In addition, more severe gram mutants also display mild homeotic conversions indicating a role of GRAM in the control of expression domains of the B and C functions. Genetic interactions between sty and gram mutants revealed common and distinct functions during vegetative and reproductive development, one aspect of which is the cooperative control of the B and C domains. We also report on an unexpected connection between STY and hormone-mediated processes, suggesting a more general role for STY in developmental control.
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Materials and methods |
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The wild-type lines JI98 (the progenitor of line 165E), JI75 and the
gram-3 mutant (Golz et
al., 2004) were kindly provided by Rosemary Carpenter (John Innes
Centre, Norwich, UK). The wild-type line Sippe 50 and the mutants sty,
phan-ambigua and gram-1 (referred to as phan and
gram, respectively) were obtained from the collection at the IPK,
Gatersleben, Germany (accession numbers MAM428, MAM316, MAM250 and MAM 146,
respectively). To reduce the influence of the genetic background the genuine
`Gatersleben' background, corresponding to the Sippe 50 line was generally
used. The 165E line was used for segregation analyses to enhance the
probability of sequence polymorphisms between mutant and wild-type
alleles.
Arabidopsis lug-1 seeds (N8031) were obtained from the Nottingham Stock Centre.
Molecular biology
Detailed information on isolation of proteins, nucleic acids, PCR primers,
PCR conditions and other methods used but not explicitly documented in this
report are available upon request.
DNA- and RNA-related methods
DNA for large scale segregation studies by PCR was prepared from 50-100 mg
of leaves, adopting a protocol developed for Arabidopsis
(Klimyuk et al., 1993) using
96-well plates. Polymorphisms were detected as CAPS (cleaved amplified
polymorphic sequences) by restriction of PCR fragments and separation on
agarose gels or as single-nucleotide polymorphisms (SNP) with the WAVE method
(Kuklin et al., 1997
).
Protein-related methods
The cDNAs of the entire GRAM protein and amino acids 173-509 of STY
(displaying the lowest degree of homology between STY and STY-L; see
Fig. 1) were cloned into the
pGEX-3X and pQE60 vectors, respectively. The recombinant proteins were
expressed in E. coli and purified by utilising the N-terminal GST
extension (for GRAM) or the C-terminal His-tag (for STY). Antisera were
produced in rabbits (Pineda Antibody Service, Berlin, Germany) and affinity
purified, in two steps, against antigens immobilised on HiTRAP NHS-activated
HP columns (Amersham Biosciences). First, most of the antibodies interacting
with the tags and nonspecific antibodies cross-reacting with plant proteins
were removed. In the second step antibodies specifically interacting with the
immobilised GRAM or STY antigens were obtained.
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Yeast two-hybrid screening
The coding region of the STY cDNA was cloned into pGBT9 and into
pBKT7. The screening procedure after library transformation followed a
published protocol (Davies et al.,
1996). For detecting ternary complexes the AmSEU3A cDNA was cloned
into the TFT vector and used as previously reported
(Egea-Cortines et al., 1999
).
Some of the screens were performed by applying the Matchmaker library
construction and screening protocol (Clontech) and used a normalised full
plant yeast expression library for mating (S. Masiero, Z.S.-S. and H. Sommer,
unpublished). For directly testing interactions in yeast, cDNAs were cloned
into pBKT7 and pGAD424.
In situ analysis of RNA and protein expression
Tissue preparation, in situ hybridisation and immunolocalisation
experiments were performed as previously described
(Davies et al., 1996;
Perbal et al., 1996
;
Zachgo et al., 1995
). The
digoxigenin-labelled STY antisense probe contained the internal, non-redundant
region of the STY cDNA (position 520 to 1520). The GRAM probe was prepared
from the full-size GRAM cDNA.
Histology and scanning electron microscopy
Histological sections were prepared and viewed according to the method of
Golz et al. (Golz et al.,
2004). For observations on the vascular skeleton leaves were
dehydrated in ethanol, cleared with NaOH and stained with basic fuchsin
(Sigma) as described previously (Fuchs,
1963
). Photographs were taken with a Leica MZ FIII microscope
using UV light. Scanning electron microscopy (SEM) with fresh freeze-fractured
leaves was performed as reported previously
(Efremova et al., 2001
).
Auxin response and polar auxin transport inhibition assays
Three-week-old in vitro cultured seedlings
(Heidmann et al., 1998) were
transferred to 0.5x MS medium
(Murashige and Skoog, 1962
)
containing polar auxin transport inhibitors or auxins. For inhibition of polar
auxin transport 0.5-20 µM 1-N-naphthylphthalamic acid and
2,3,5-triiodobenzoic acid (NPA and TIBA, respectively, both from Duchefa
Biochemie BV, Holland) were used as described previously
(Mattsson et al., 1999
). For
auxin response assays, indoleacetic acid and 2,4-dichlorophenoxyacetic acid
(IAA and 2,4-D, respectively, both from Sigma) were dissolved in 1 M NaOH and
in DMSO, respectively, and were added at 0.5-6 µM to plant growth
media.
For measurement of polar auxin transport
(Okada et al., 1991) the upper
end of 2.5 cm long inflorescence stem segments, adjacent to the oldest flower
of 8-weeks-old plants, were submerged in 0.5x MS medium containing 1.45
µM IAA and 4.8 nCi/30 µl [3H]IAA (Amersham). After incubation
for 16 hours, the opposite 5 mm end of the segments was excised, the
radioactivity extracted for 12 hours in 1 ml ethanol and measured in a
Beckmann LS-6500 liquid scintillation counter. Segments with the basal end
submerged (with movement in the physiological direction) were used as
controls.
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Results |
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RT-PCR or northern blot analysis revealed that the sty transcript in sty mutants is reduced by up to 90% when compared to wild type (not shown), possibly because of an instability of the mutant mRNA.
GRO/TUP1-like co-repressors in Antirrhinum
There is a high degree of amino acid sequence conservation between LUG and
STY with both proteins having an N-terminal LUFS domain, followed by a
glutamine-rich domain, a variable region and a C-terminal 7 WD repeat domain
(Fig. 1)
(Conner and Liu, 2000). STY
differs from LUG in having slightly shorter glutamine-rich domains and an
additional WD repeat. This domain structure is similar to GRO/TUP1-like
proteins in Drosophila, mammals and yeasts
(Conner and Liu, 2000
).
A second STY-like (STY-L; EMBL accession AJ620906) cDNA
was identified in the Antirrhinum EST collection. STYL
appears to be the orthologue of LUH in Arabidopsis and
displays a similar degree of amino acid sequence divergence from STY as that
reported between LUG and LUH (Conner and
Liu, 2000). These include a large deletion within the N-terminal
glutamine-rich domain (Fig. 1) and conservation of amino acids that distinguish LUH from LUG (not shown). The
functional consequences of these deviations are not known, but our studies in
yeast suggest that STY and STY-L differ slightly in the range of proteins with
which they can interact (Table
1, and not shown). Similarly, two structurally closely related
TUP1-like proteins differing by internal deletions are present in fission
yeast and play partially redundant roles in transcriptional regulation
(Hirota et al., 2003
;
Janoo et al., 2001
). In
support of such redundancy, interaction between STY and STY-L can be observed
in yeast (Table 1).
|
Using STY as bait, several transcription factors were identified from a
screen of about 5x107 yeast recombinants in various
two-hybrid screens (Table 1;
see Materials and methods). A major group of interactors included four
proteins with sequence similarity to SEUSS (SEU) in Arabidopsis,
which we called AmSEU. SEU is a putative co-repressor that interacts both
genetically and physically with LUG
(Franks et al., 2002). Our
studies in yeast suggest that interaction between STY and AmSEU facilitates
formation of higher order complexes with other proteins
(Table 1).
A second major group of proteins that interact with STY in yeast belong to
the YABBY family of transcription factors (GRAM, PROL and AmINO; see
Table 1). YABBY genes
were first identified in Arabidopsis
(Sawa et al., 1999b;
Siegfried et al., 1999
) and
form a small gene family of six members. In Antirrhinum, there are
only five YABBY genes, with GRAM being the only orthologue
of two closely related genes FILAMENTOUS FLOWER (FIL) and
YAB3 (Golz et al.,
2004
). Two other YABBY proteins, AmYAB2 and AmCRC, do not interact
with STY, but AmCRC can form a higher order complex with STY and AmSEU
(Table 1). In agreement with
their structural and functional similarity to the Antirrhinum
proteins STY and GRAM we found that the Arabidopsis protein LUG
interacts with FIL and YAB3 in yeast.
The YABBY proteins identified in yeast screens all contained the N-terminal zinc-finger and the internal variable region, but in many instances lacked the YABBY-domain and the C-terminal region. This suggests that the internal variable region or the zinc-finger domain represent the region interacting with STY.
Based on the synergistic genetic interaction between sty and either cho, fis or des, we expected that some of the STY-interactors might be the proteins encoded by FIS, CHO or DES. However, CAPS markers developed for the four AmSEU genes and STY-L did not co-segregate with cho, fis or des.
Genetic interaction between STY and GRAM in the control of flower development
sty gram double mutants were generated and their phenotypes
compared to the single mutant lines to test possible interactions in vivo.
The subtle homeotic defects of gram
(Golz et al., 2004)
(Fig. 2B,E) and sty
flowers (Motte et al., 1998
)
(Fig. 2C,F) are dramatically
enhanced in the sty gram double mutant
(Fig. 2G-I). The whorled
organisation of sty gram flowers is often disrupted, making it
difficult to assign floral organs to a particular whorl
(Fig. 2H). Most often the
dorsal and the two ventral sepals in the first whorl are petaloid and second
whorl organs are narrow, radialised or stamenoid
(Fig. 2H). Stamens in the third
whorl can be sterile or feminised. Carpels in the fourth whorl are misshapen
with a broadened basal part resembling the gynoecium of sty and a
short, sometimes narrow and split style similar to styles in gram
flowers. sty gram inflorescences and flowers display several other
defects such as delayed flower formation, retarded flower development and
frequent abortion, resulting in irregular inflorescences (not shown).
Furthermore, floral organs can be filamentous and their number reduced
(Fig. 2I), in extreme cases to
two sepals, two narrow radialised petaloid structures and a rudimentary
gynoecium (Fig. 2F). The
severity and range of defects were similar in all genetic backgrounds.
|
STY and GRAM co-operate in the control of vegetative development
Vegetative development of sty gram double mutants is severely
disturbed, with irregular internode length, aberrant phyllotaxis, partial
fusion of the cotyledons and arrested growth of seedlings
(Fig. 3B,C). The shoot apical
meristem is still functional in these seedlings, as spontaneous bursting, or
manual disruption of the fused region results in organ formation and growth.
None of these defects is revealed by gram or sty single
mutant plants, but sty in the JI98 background and gram-3 or
gram in the JI75 background show some of the anomalies, although in a
less severe form.
|
Taken together, these observations suggest that STY genetically interacts with GRAM for initiation and positioning of primordia and in the control of leaf polarity and growth.
Defects in sty mutant vascular development
The observation that sty influences the leaf phenotype of
gram prompted us to study sty leaf morphology in detail.
Measurement of the overall length and width of mature lower leaves did not
reveal obvious differences compared to wild type, although segregating
populations sty plants more often bear smaller or slightly narrower
leaves at upper nodes than wild-type sisters, reminiscent of the narrow leaves
of lug mutants (Liu and
Meyerowitz, 1995; Liu et al.,
2000
). Interestingly, however, the venation pattern of
sty leaves differ from wild type in that the major (primary and
secondary) veins are slightly broader and the density of minor veins at the
tip of the leaf is reduced (Fig.
4C,G). Furthermore, vascular strands are not properly aligned
(insets in Fig. 4F-G). The
severity of these defects is variable, ranging from near wild type to very
aberrant. Thus, unexpectedly, STY has a role in vascular
development.
|
STY and GRAM expression patterns indicate early overlap and late exclusion
The observed physical and genetic interactions between STY and
GRAM suggest that these genes have overlapping expression patterns.
The precise site and time of this overlap during vegetative and reproductive
development was determined using both in situ mRNA hybridisation and protein
immunolocalisation. The pattern of protein and RNA accumulation is similar for
each gene (Fig. 5A-D) and
therefore we arbitrarily chose either RNA or protein pattern for
documentation.
|
|
In summary, potential physical interactions between GRAM and STY are limited to a short period when both genes are expressed in the same cells of initiating vegetative and floral organ primordia. Expression of both genes becomes mutually exclusive later in development.
The STY and GRAM proteins localise to the nuclei as indicated by quenching of fluorescence at sites of protein expression following a DAPI treatment (Fig. 5E-F). Intriguingly, a considerable proportion of the GRAM protein remains in the cytoplasm. This does not appear to be an artefact because cytoplasmic signals cannot be detected in the gram-3 mutant (Fig. 7B) or in tissues where GRAM is not expressed.
|
Organs of phantastica (phan) mutants grown at 20-22°C
show varying degrees of abaxialisation, whereas at 16-17°C organs are
radially symmetric and almost completely lack adaxial identity
(Waites and Hudson, 1995;
Waites et al., 1998
).
Examining the STY expression pattern in phan mutants
therefore should reveal to what extent STY is regulated by PHAN
and/or by adaxial cell identity. Initiation of STY expression, and its early
restriction to the adaxial region does not depend on PHAN as P4/P5
phan primordia express STY within their adaxial region
(Fig. 7C,D). Furthermore,
residual ad-abaxial asymmetry of abaxialised organs initiated at 17°C is
still reflected by an adaxial STY pattern
(Fig. 7C). Thus, polarised
STY expression in phan mutant organs is not sufficient to
promote adaxial cell identity, although STY expression in
gram-3 organs appropriately responds to polarity.
Hypersensitivity of sty mutants towards polar auxin transport inhibitors and exogenously applied auxins
Alterations in the leaf venation pattern, mild problems with phyllotaxis
and the tendency of sty and sty gram mutants to fasciate
(not shown) suggest that there may be local changes in auxin levels, responses
or movement (Mattsson et al.,
2003; Okada et al.,
1991
; Sieburth,
1999
). To investigate this aspect of the STY function we
applied auxin transport inhibitors and exogenous auxins to wild-type and
sty seedlings.
Low (0.5-1 µM) concentrations of the auxin transport inhibitor NPA (or
TIBA, not shown) hardly affect wild-type morphology but induce a dramatic
change in sty seedlings (Fig.
8E). Instead of the main shoot composed of leaf-bearing
internodes, sty seedlings develop a pin-like structure. Lateral
shoots initiate from the hypocotyl after several weeks of growth and produce
leaves with very broad midveins, comparable to leaves of wild-type plants
grown on 10-20 times higher NPA concentration (not shown). Growth and
elongation of sty roots are also more severely affected by NPA than
those of wild-type seedlings (Fig.
8B,E). In fact, the roots of sty control seedlings are
already shorter than wild-type ones and grow in an agravitropic manner (not
shown), reminiscent of the behaviour of Arabidopsis pin2 and
pin3 mutant seedlings (Friml et
al., 2002; Müller et al.,
1998
). The response of Arabidopsis lug-1 seedlings to
treatment with NPA is similar to sty (not shown).
|
Hypersensitivity towards auxins and polar auxin transport inhibitors can result from reduced auxin transport. Indeed, transport measurements indicate a 20-30% inhibition of polar auxin transport in sty mutants compared to wild type (Fig. 9).
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Discussion |
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The LUG and STY proteins share similarities in domain structure with
GRO/TUP1-like co-repressors (Conner and
Liu, 2000). Members of the GRO/TUP1 super-family cannot directly
bind DNA and are recruited to the site of their function by DNA-binding
proteins, sometimes mediated by additional `adaptor' proteins such as Ssn6
(Smith and Johnson, 2000
).
Subsequent recruitment of histone deacetylases results in transcriptional
silencing (Flores-Saaib and Courey,
2000
). Finding proteins interacting with STY is therefore an
approach that is likely to identify proteins required for the formation of
repressor complexes. Using a yeast two-hybrid screen we found that several
members of the YABBY family interact with STY. Proteins in this group
containing the YABBY domain (Bowman,
2000
), a partial HMG-box with no DNA-binding specificity
(Kanaya et al., 2002
).
Interestingly, interaction between GRO/TUP1-like co-repressors and HMG-domain
proteins is a common feature in mammals, Drosophila and yeast
(Brantjes et al., 2001
;
Cavallo et al., 1998
;
Deckert et al., 1995
),
although the HMG-domain proteins involved are quite diverse. It has been
suggested that HMG proteins are architectural factors that are necessary in
combination with GRO-like co-repressors and other proteins to form a
'repressosome' (see Introduction), a function that might also be associated
with STY/YABBY complexes in plants. Indeed, the function or stability of the
protein complex that contains STY/GRAM may well depend on additional
components, which would fit with the influence of `background factors' on the
phenotype of sty and gram mutants.
By analogy to the GRO/HMG-box proteins, it is likely that the diverse functions of STY and GRAM arise from acting together as well as separately in complexes with other proteins. A further complication results from the possible partial redundancy of the STY function with STY-L, and the GRAM function with other members of the YABBY family. Despite this, developmental events in which STY and GRAM are likely to interact physically in vivo will be discussed below, along with independent STY and GRAM functions.
STY and GRAM in the control of floral organ identity
Several lines of evidence support the function of a STY/GRAM complex in the
control of floral organ identity. Firstly, the mild floral homeotic defects in
control of the outer boundary of the floral B and C functions in sty
and gram mutants indicate an overlap of their function suggesting
that the two genes act in the same control pathway. We assume that incomplete
functional equivalence of redundant factors, or incomplete overlap of their
expression patterns is responsible for the weak defects displayed by the
single mutants. In support of this, gram prol double mutant flowers
display a greater degree of homeotic conversions than gram single
mutants (J.F.G., unpublished). Similarly, two TUP1-like proteins in fission
yeast have partially redundant roles in chromatin remodelling and
transcriptional repression (Hirota et al.,
2003). Secondly, the combined loss of STY and
GRAM results in more severe homeotic conversions than loss of either
STY or GRAM alone. This is consistent with the idea that
eliminating two components of a protein complex is more deleterious than
eliminating just one. Third, incipient floral organs in the outer whorls
concomitantly express STY and GRAM at early developmental
stages, prior to establishment of the B and C functions
(Bradley et al., 1993
). Later
this overlap resolves in a complementary pattern suggesting that the proteins
perform functions other than together controlling organ identity.
Enhanced expansion of the C function to the outer whorls in lug
fil double mutant compared to single mutant flowers has also been noted
(Chen et al., 1999). The
control of the C domain by the STY/GRAM and LUG/FIL complexes thus appears to
be conserved between Arabidopsis and Antirrhinum, as do the
respective protein interactions observed in yeast. Interestingly, several
abnormal features of Arabidopsis lug or fil single mutant
flowers, such as filamentous organs, reduced organ number and aberrant whorl
organisation (Chen et al.,
1999
; Liu and Meyerowitz,
1995
; Sawa et al.,
1999a
) are not revealed in sty or gram single
mutants, but are in the sty gram double mutant. These differences
suggest deviations in the range of control events exerted by the respective
proteins or protein complexes in the two species.
Expansion of the B and C domains occurs concomitantly suggesting that the
two control processes are linked. This may indicate that both the B and C
control genes are regulated by STY/GRAM. Testing the physical association of
STY/GRAM with regulatory regions of class B and class C genes will resolve
whether this repression is direct. An alternative is that STY/GRAM govern
processes preceding organ identity control, such as the timing of organ
initiation or positioning of primordia, as discussed below. In accord with
this idea, ectopic expansion of the C-function gene PLENA
(PLE) in sty flowers is preceded by changes in expression of
several other floral control genes suggesting that PLE is not the
only target of STY regulation
(Motte et al., 1998).
STY and GRAM in the control of organ initiation
Impaired initiation and positioning of leaves (aberrant phyllotaxis) and
floral organs (lack of whorled organisation) is one of the severe changes
during development of the sty gram double mutant. Since the two
proteins are co-expressed in the nuclei of lateral organ primordia their
interaction in the control of organ initiation is feasible. The fact that the
single mutants do not display severe developmental defects in this process is
perhaps due to redundancy. Positioning and emergence of lateral organs are
controlled by the plant hormone auxin
(Reinhardt et al., 2003).
Given the observed interaction between STY and hormone-mediated
control processes, as discussed below, it is possible that enhanced
phyllotaxis defects in the sty gram mutant are related to impaired
auxin perception or movement.
STY in the control of leaf polarity
The sty mutant does not display obvious loss of organ polarity,
perhaps because of redundancy with the STY-L gene. However, two
observations suggest a redundant role for STY in the control of
adaxial fate. Firstly, STY expression becomes adaxially restricted
similar to Arabidopsis genes such as PHABULOSA
(PHB) that promote adaxial identity in lateral organs
(McConnell et al., 2001). This
restriction of STY occurs in P4 primordia, subsequent to adaxial
restriction of AmPHB during late stage P1
(Golz et al., 2004
) and thus
STY is not likely to be involved in the initial establishment of
adaxial asymmetry. Consistent with this, abaxialised phan mutant
primordia retain spatially correct adaxial STY expression, suggesting
that asymmetric STY expression is independent of PHAN.
Nevertheless, STY expression expands into the adaxialised margins of
gram-3 organs, indicating that STY expression can follow
adaxial fate. Secondly, the radialised needle-like leaves that develop in the
gram sty double mutant suggest a common role of GRAM and
STY in leaf asymmetry, supported by co-expression of the two genes.
However, gram sty needles, unlike the abaxialised needles
occasionally forming in gram mutants, show both abaxialised and
adaxialised characters. The reason for this is presently not clear and the
role of STY in the control of adaxial identity remains enigmatic.
Independent roles of STY and GRAM in the control of leaf lamina growth
Clonal analysis suggests that GRAM promotes cell divisions in
marginal cells of leaf primordia. In the gram mutant growth at the
margins is reduced, but the effect on leaf width is in part compensated by
enhanced cell divisions in more central regions
(Golz et al., 2004). Reduction
of sty gram leaf width indicates that STY is needed for
compensatory growth and therefore that STY might control
proliferation in the central regions of the wild-type leaf. In accordance with
this, STY is expressed during the phase of expansion in the vascular
tissue and at the junction between abaxial and adaxial regions of young
leaves. The role of STY in promoting growth weakly manifests in
reduced leaf width in the sty mutant, perhaps because enhanced cell
divisions in the margin replace cells derived from more central regions.
Reduced width of sty gram leaves might thus reflect independent roles
of GRAM and STY in the control of lateral growth.
STYLOSA in the control of venation and hormone-mediated processes
A function of STY during vascular development is underscored by
prominent expression of the gene in provascular and vascular cells, and is
confirmed by impaired venation and changes in auxin responses/polar auxin
transport in the sty mutant. In contrast, GRAM is not
expressed in provascular cells and gram mutants just weakly respond
when grown on auxins or auxin transport inhibitors (N.E., unpublished).
Aberrant venation in gram might be the consequence of polarity and
growth defects, as these features are tightly linked
(Dengler and Kang, 2001;
Waites and Hudson, 1995
).
Thus, the enhancement of vascular defects in sty gram results from
the combined effects of both genes.
Our observations on impaired development of major and minor leaf veins and
enhanced responses of the sty mutant to auxins or polar auxin
transport inhibitors strongly suggest a role of STY in producing or
mediating hormone-dependent signals. The precise site at which STY is
involved in auxin signalling or transport is not clear. Transport measurements
suggest partial inhibition of polar auxin transport, but give no hint whether
this is due to a direct effect of STY on transport or an indirect effect of
other STY-controlled events. Since most hormone-related processes are
interconnected (Coenen and Lomax,
1997; Ephritikhine et al.,
1999
) it is possible that other hormones are primarily influenced
by STY.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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