School of Biological Sciences, Monash University, Melbourne, VIC 3800, Australia
Author for correspondence (e-mail:
david.smyth{at}sci.monash.edu.au)
Accepted 11 May 2004
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SUMMARY |
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Key words: PETAL LOSS (PTL), Arabidopsis, Trihelix, GT-factor, Flower development, Perianth
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Introduction |
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A range of genes that regulate floral architecture has been identified in
Arabidopsis thaliana. The PERIANTHIA (PAN) bZIP
transcription factor gene ensures that flowers arise with the appropriate
numbers of organs in the outer three whorls (four sepals, four petals and six
stamens) (Chuang et al., 1999).
In pan mutants there are usually five in each case. By contrast, the
architecture of the second and third whorls is supported by the UNUSUAL
FLORAL ORGANS (UFO) F-box gene
(Durfee et al., 2003
;
Laufs et al., 2003
). Another
gene, PRESSED FLOWER (PRS), encoding a homeodomain protein,
has roles in defining the lateral regions of the flower primordium rather than
the radial regions (Matsumoto and Okada,
2001
). In prs mutants, lateral sepals are frequently
absent.
Other genes function to define boundaries. For example, the
SUPERMAN (SUP) zinc finger gene acts to control cell
proliferation in the boundary between the stamens and carpels
(Sakai et al., 1995). In
addition, boundaries between individual organs are controlled by CUP-SHAPED
COTYLEDON (CUC) genes encoding NAC transcription factors. These act to keep
the primordia of adjacent floral organs, especially the sepals, separate
(Aida et al., 1997
).
Genes required for the development of specific organ types have also been
described. For example, another function of UFO is to promote petal
outgrowth, perhaps by targeting an inhibitor of this process for degradation
(Durfee et al., 2003;
Laufs et al., 2003
). Another
gene that specifically promotes petal growth, RABBIT EARS
(RBE), encodes a zinc finger protein
(Takeda et al., 2004
). In null
rbe mutant plants, petals are mostly filamentous or absent.
The PETAL LOSS (PTL) gene of Arabidopsis plays a
unique role in controlling perianth development
(Griffith et al., 1999). In
mutant plants, sepals are mis-shapen and sometimes fused with an adjacent
sepal. Petals are often absent, and their mean number per flower falls
progressively so that later-formed flowers usually have none. Those petals
that do arise are often smaller than normal and are sometimes trumpet-shaped.
Petal primordia occupy the same regions of the mutant flower primordium as in
the wild type (internal to each of the inter-sepal zones), although the four
regions are somewhat enlarged. Also, their initiation may be delayed. The
number of petals per flower is influenced by the presence of a dominant allele
of the PETAL LOSS MODIFIER (PMD) gene, with more petals
arising per flower in pmd-1d background.
Griffith et al. proposed that PTL normally functions to support the action
of a petal initiation signal (Griffith et
al., 1999). This was proposed to act in four regions of the
developing flower, internal to the inter-sepal zones. These regions might be
enlarged in ptl mutants such that response to the signal is weakened
and the threshold is only occasionally reached. It was also proposed that
sensitivity to the signal is boosted in the pmd-1d background.
The orientation of petals within the flower is also disrupted in
ptl mutants. Some face sideways, and others are reversed. Griffith et
al. suggested that the response to another signal, acting with defined
polarity within the flower primordium, was being disrupted in ptl
mutants, such that petal primordia sometimes adopted an inappropriate, or even
default, orientation (Griffith et al.,
1999).
In this study, we report the identification of PTL as a transcription factor gene of the plant-specific trihelix family. PTL is the first member of this family known to control morphogenesis others known to date are associated with the regulation of light-responsive genes. PTL is expressed in four zones between newly arising sepals, where it may help to maintain their separation. Consistent with this idea, ectopic expression of PTL results in growth inhibition. In addition, some fusion of adjacent sepals occurs in ptl mutant plants. Surprisingly, PTL is apparently not expressed in developing petal primordia. This suggests that the disruptions to petal initiation and orientation in ptl mutants are caused indirectly, perhaps as a consequence of overgrowth in the nearby inter-sepal zones.
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Materials and methods |
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Plants were grown at 20-25°C in natural daylight supplemented with
continuous Cool White fluorescent light. Stages of flower development follow
Smyth et al. (Smyth et al.,
1990).
Cloning of PETAL LOSS and isolation of cDNA
Clones of Yeast Artificial Chromosomes (YACs) and TAMU Bacterial Artificial
Chromosomes (BACs) for generating contigs in the region overlapping
PTL were obtained from The Arabidopsis Biological Resource Center.
PTL was localized to a 10.7 kb XbaI genomic fragment present
in the right end of BAC T24M12 (Columbia ecotype). This was cloned into
pBluescript SK(+) (Stratagene) for sequencing, or into the binary vector
pBIN19 for complementation tests. In the latter, the construct (D289) was
transferred to Agrobacterium tumefaciens strain AGL1, and then into
ptl-1 mutant plants by bacterial infiltration of flower buds.
A cDNA library, made from Ler inflorescence tissue with buds up to
stage 12 using ZAPII (Stratagene) (provided by Detlef Weigel, Salk
Institute), was probed with BAC T14K22, and the positive clones probed in turn
with the right end XbaI subclone of BAC T24M12. One positive clone
(D171) was obtained (GenBank Accession number AY555728).
Nuclear localization
PTL coding sequences in cDNA clone D171 were translationally fused
to the C terminus of the GFP coding sequence from pBIN mGFP5-ER (with ER
deleted). This was inserted into plasmid pART7
(Gleave, 1992) downstream of
the 35S CaMV promoter sequence. Transient expression of 35S::GFP-PTL in onion
epidermal cells, and GFP fluorescence, followed the protocol of Weigel and
Glazebrook (Weigel and Glazebrook,
2002
).
Analysis of PETAL LOSS RNA
RT-PCR followed the OneStep procedure (Qiagen) starting with total RNA
extracted using the RNeasy procedure (Qiagen). Primers for transcripts of
PTL and the two control genes APETALA3 (AP3) and
ACTIN2 (ACT2) were designed to flank or overlap introns,
generating cDNA products of 738 bp (PTL), 356 bp (AP3), or
1,153 bp (ACT2). Each reaction started with approximately 0.5 µg
total RNA, and the PTL and AP3 reactions were identical
(55°C annealing temperature with 20 seconds extension for 30 cycles)
except that `Q solution' (Qiagen) was added to the PTL reaction. The
ACT2 reaction conditions were 52°C, 30 seconds and 28 cycles.
In situ hybridization, using digoxigenin-labelled sense and antisense
probes made using the cDNA plasmid D171, essentially followed the protocol of
Heisler et al. (Heisler et al.,
2001).
Generation and analysis of GUS reporter constructs
The PTL regulatory region was translationally fused with the
uidA gene of E. coli encoding ß-glucuronidase (GUS).
The BAC clone T14K22 (Columbia genomic DNA) supplied the sequences used,
either restriction fragments or PCR products. These were ligated into the
shuttle vector pRITA (Eshed et al.,
1999; Eshed et al.,
2001
) using an NcoI site present at the first methionine
codon of GUS. After sequencing to confirm appropriate cloning, the pPTL::GUS
casettes were cloned into the NotI site of the binary vector pART27
(Gleave, 1992
) (conferring
kanamycin resistance in plants), or a Basta resistance derivative of this,
pMLBART. These were then transferred into Columbia plants as before.
Five PTL reporter constructs were made, three with the first exon and intron (p8.0i::GUS, p2.0i::GUS and p1.3i::GUS), and two without (p8.0::GUS and p2.0::GUS). For the first three constructs, the fusion involved either the first methionine (p2.0i) or the first tyrosine (p8.0i and p1.3i) of the second exon. For the last two (p8.0 and p2.0), the fusion was at the second methionine of the first exon. Where PCR had been used for cloning, full sequencing revealed one change in both p8.0 and p8.0i (one less T in a string of 15 located 4,957 bp upstream of the first methionine), and 9 base substitutions in p2.0i. The latter did not change the translated sequence, and as the expression pattern for p2.0i was closely similar to that of the error free p1.3i, it was assumed the changes had had no functional consequences.
GUS staining of transformed lines was carried out by briefly fixing material in 90% acetone, staining overnight in 2 mM X-Gluc in 50 mM phosphate buffer (pH 7.2) at 37°C, and then removing chlorophyll using ethanol. Most staining solution also included 6 mM potassium ferri- and ferro-cyanide to reduce bleeding. Tissues were examined as whole mounts in 70% ethanol, or were embedded in Paraplast Plus, sectioned at 8 µm, and viewed using dark-field optics. The precipitated product of the ß-glucuronidase reaction appears blue in bright field, but pink in dark field.
Ectopic expression of PTL using the lac repressor/operator transactivation system
In this system (Moore et al.,
1998), a driver line carries a promoter of choice driving a hybrid
transcription factor (the lac repressor translationally fused with
the GAL4 activation domain, LhG4). The target line carries the
lac operator (pOp) in tandem copies upstream of a gene of interest.
Expression of the target gene is induced in progeny of crosses between the two
lines, here indicated as pDRIVER>>TARGET
(Eshed et al., 1999
;
Eshed et al., 2001
).
Two PTL driver constructs were made containing either 1.3 kb upstream of
the first methionine codon, the first exon and the intron [pPTL(1.3i)], or 2.0
kb of the upstream region [pPTL(2.0)]. These PTL regulatory sequences
were each inserted into the modified shuttle vector pBJ36-LhG4
(Eshed et al., 2001), and then
into the NotI site of pMLBART. ptl-1 plants were
transformed, and 11 independent insertion lines carrying pPTL(1.3i), and seven
of pPTL(2.0) were recovered. Their expression was assessed by intercrossing
with a pOp::GUS target tester line (provided by Yuval Eshed, University of
California Davis), and the patterns closely matched those of comparable
pPTL::GUS translational fusion lines. Three other driver lines, carrying
either 1.7 kb of the APETALA1 upstream promoter region (pAP1)
(Emery et al., 2003
), or 0.42
kb of the APETALA3 promoter region (pAP3), or the CaMV 35S control
region present in 1.35 kb of pART7 (p35S)
(Gleave, 1992
), were also
provided by Yuval Eshed.
A PTL target construct was made by inserting the PTL coding
sequence from cDNA clone D171 downstream of pOp present in plasmid p12OpBJ36
(Eshed et al., 2001). The
insert was excised with NotI and inserted into pART27. Four
independent target insertion lines were obtained in ptl-1 background,
and 12 in Columbia background.
Scanning electron microscopy (SEM) was carried out as described previously
(Griffith et al., 1999).
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Results |
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A cDNA covering this site was isolated from an inflorescence library, and the candidate gene shown to correspond to PTL, in that a 10.7 kb genomic clone encompassing the gene (Fig. 2B) fully complemented the ptl-1 mutant phenotype. Also, all five mutant alleles (of similar phenotype) were associated with sequence changes that are likely to result in loss of function of the deduced polypeptide (Fig. 2A).
|
The deduced PTL protein (Fig.
2A) contains a long helical region between the two trihelices that
is conserved in family members closely related to PTL
(Nagano et al., 2001). The
center of this is predicted to form a coiled-coil
(Fig. 2A), possibly associated
with multimer formation. In addition, there is a serine-threonine rich region
and a glycine rich region, each commonly found in transcription factors. There
are also several putative nuclear localization signals
(Dehesh et al., 1995
), and we
have shown that the PTL protein accumulates in the nucleus
(Fig. 2C,D).
PTL is expressed at relatively low levels
We were unable to detect PTL transcripts by northern
hybridization, so we screened tissues using RT-PCR
(Fig. 3). In comparison with
AP3, a gene strongly and specifically transcribed in floral tissues
(Jack et al., 1992),
PTL is weakly expressed. The highest level was found in
inflorescences (including flowers up to stage 12). Relatively high expression
was also seen in 7-day-old seedlings, although not in seedlings one day
younger. AP3 transcripts also increased markedly in seedlings between
6 and 7 days, suggesting that inflorescences had commenced development. Lower
levels of PTL transcription were detected in the other tissues
examined (Fig. 3).
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PTL is also expressed in lateral regions of developing floral organs
Expression of PTL was also seen fully around the edges of
developing sepals (Fig. 4A,D-H)
from stage 5 when they enclose the developing flower meristem. It extended
several cells inward from the epidermis, particularly in adaxial regions at
the sepal base, persisting until around stage 9.
Petal primordia arise between the sepals and internal to them at stage 5. It was not possible to determine whether the few petal-initiating cells carried GUS product at this stage, but, surprisingly, it was not observed in the small developing primordia at stages 6-7 (Fig. 4I,J). GUS product was first detected at around stage 8, just before petal primordia commence rapid growth (Fig. 4K). Even then, it was present only basally in the flanks, and this faded by stage 13 when petals were fully grown.
GUS product was also present in lateral regions of stamen primordia, specifically at the top of the filament and the base of the anther, from the time they differentiate from each other at stage 7 (Fig. 4L) until stage 9.
PTL is also expressed in margins of developing leaves and in some other vegetative tissues
In seedlings, PTL expression was first seen in developing leaves
(Fig. 4M,N), commencing only
after their primordia had extended over the shoot apical meristem. It was
first present in a continuous band around their perimeter, extending through
all three cell layers (Fig.
4N). Later, expression became limited to the basal region,
including the petiole (Fig.
4M). Strong GUS staining was also seen in stipules throughout
their life (Fig. 4N).
Weaker PTL expression was seen in the axils of cauline leaves and floral pedicels, with the intensity of GUS staining increasing as individual axils aged (Table 2). Staining intensity was somewhat lower on average in the shorter promoter lines, p2.0i::GUS and p1.3i::GUS, than in p8.0i::GUS lines. Some other vegetative regions were also stained, although relatively weakly (Table 2). These included the basal node of the rosette, the floral receptacle, and the vasculature of the root.
Overall, localization of the GUS reporter gene product is consistent with RT-PCR results (Fig. 3). Furthermore, PTL expression in ptl mutant plants, and in pmd-1d (Ler) plants, was not noticeably different from the wild-type pattern in either case.
The intron is required for expression in flower primordia, but not in sepal and leaf margins
A striking difference in staining pattern was seen in reporter lines when
the first exon and intron were absent (p8.0::GUS and p2.0::GUS)
(Table 2). Staining in the
flanks of flower primordia, and subsequently in the four inter-sepal zones,
could not be detected (Fig.
4C). Staining in basal regions of developing petals was also
absent. Thus, the intron is required to drive expression in these regions. (An
involvement of exon 1 sequences is possible although regulatory sequences
rarely occur in translated regions.) By contrast, staining around the margins
of sepals and leaves was similar with or without the exon and intron
(Fig. 4C; Table 2).
Expression in developing stamens was present without the intron, but only in the 8.0 kb construct (Table 2). It is apparently controlled by redundant elements present both in the intron and upstream of 2.0 kb. The intron also contains elements that dampen PTL expression in other vegetative regions (Table 2).
Early expression in flower primordia is required to complement the ptl mutant phenotype
In ptl mutants, visible defects were limited to sepals and petals
(e.g. Fig. 5A). To test which
components of the PTL expression pattern are associated with these
defects, we carried out complementation tests using expression lines with or
without the intron (i.e. with or without expression in the early flower
primordium and inter-sepal zone). To do this, we used the two-component system
of Moore et al. (Moore et al.,
1998) (see Materials and methods). Two sets of driver lines
containing PTL regulatory sequences (one set with the intron
[pPTL(1.3i)], and one without [pPTL(2.0)], were each crossed with four
replicated target lines containing the PTL coding sequence, all in
ptl-1 mutant background. [Although the latter driver carries an
additional 0.7 kb of the upstream region (1.3 to 2.0 kb), no
controlling elements were identified in this region
(Table 2).]
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Interestingly, in six of the 11 combinations in which the intron was present in the driver [pPTL(1.3i)>>PTL], a new defective flower phenotype was seen (Fig. 5C). The severity of this phenotype was correlated with the strength of expression of each driver line (previously assessed by crossing them with a GUS target line). The phenotype was characterized by narrow sepals and sometimes loss of those in lateral positions (Fig. 5E, compared with 5D). The number of petals was usually restored to four, although they were often narrow and sometimes fused to each other at the base in lateral positions within the flower. Such fusion may be the consequence of petal primordia arising much closer than normal, associated with the absence of a lateral sepal (see stage 4 bud in Fig. 5E). The two lateral stamens were also often absent. This new floral phenotype was apparently controlled by regulatory sequences within the intron because similar floral defects were not seen in any of the pPTL(2.0)>>PTL combinations. One explanation is that overexpression of PTL in the flanks of the floral meristem and the four inter-sepal zones inhibits growth of these regions. To test this, we overexpressed PTL in other defined regions of the flower meristem.
Misexpression of PTL in other tissues results in inhibition of their growth
The APETALA1 (AP1) gene is expressed predominantly in
developing flower primordia from stage 1, becoming limited to sepal and petal
primordia as they arise at stages 3 and 5
(Mandel et al., 1992). When an
AP1 driver line was crossed with another 12 independent PTL target
lines, the pAP1>>PTL progeny revealed a spectrum of floral defects
(Fig. 5F). For the most
severely affected target line (#10), plants developed normally until the
production of the inflorescence meristem. Flower primordia arose continuously,
but their development was arrested at around stage 2
(Fig. 5F). In less severely
affected target lines (e.g. #7), flower primordia were also arrested but some
filamentous floral organs, mostly carpelloid, eventually arose.
A second floral gene was also tested. AP3 drives expression
specifically in the petal and stamen sectors of flower primordia from stage 3
(Jack et al., 1992). An AP3
driver line was crossed with the same 12 PTL target lines. Strikingly, in the
resulting pAP3>>PTL plants, petal and stamen growth was often abolished
(Fig. 5G,H), or was reduced to
thin, filamentous outgrowths. In addition, the lateral sepals were often
narrow and stunted (AP3 is expressed in lateral sepal margins). The
severity of the defects across the 12 target lines was strongly correlated
with those seen using the AP1 driver (e.g. strongest in each case using line
#10, intermediate with line #7). Interestingly, the strongest phenotype
closely matched that seen when the diphtheria toxin gene DTA was
expressed by the AP3 promoter, ablating the tissues involved
(Day et al., 1995
). The weaker
effect was similar to that seen in Brassica napus flowers when cell
division was inhibited by AP3 driven expression of ICK1, an
inhibitor of cyclin-dependant kinase (Zhou
et al., 2002
).
The consequences of mis-expression of PTL more generally through the plant were tested using the CaMV 35S promoter. When the strongly responsive target line #10 was crossed with a 35S driver line, the p35S>>PTL progeny never developed beyond the production of a very short root (Fig. 5I). Using a less responsive target line (#7), progeny were arrested soon after the cotyledons emerged.
Thus it seems that strong expression of the PTL gene generally results in inhibition of growth of any tissue in which the expression occurs.
Floral expression of PTL is modified in mutants of the PINOID auxin signalling gene
Sepal development is severely disrupted in mutants of the PINOID
(PID) gene (Bennett et al.,
1995). This gene encodes a protein kinase associated with auxin
signalling (Christensen et al.,
2000
). In pid mutant flowers, sepals can be fused
laterally with each other, are irregularly spaced, and are variable in number
and size (Fig. 6A). The first
whorl arises initially as a ring of tissue without inter-sepal zones, and
sepal primordia develop irregularly from its rim much later than normal
(Bennett et al., 1995
).
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Discussion |
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PTL does not itself define the sites of origin of the four sepals, as four
still arise in appropriate positions in the absence of PTL function. Other
genes are apparently involved. One of these may be PINOID that
encodes a serine/threonine kinase implicated in auxin signalling
(Christensen et al., 2000),
possibly through its positive regulation of polar auxin transport
(Benjamins et al., 2001
).
PID is expressed in sepal primordia as they arise and grow, but not
between them (Christensen et al.,
2000
). Recently it has been shown that auxin is involved in
defining the sites of initiation of organ primordia
(Reinhardt et al., 2003
).
Thus, sepal initiation may be promoted by the localized concentration of auxin
acting through PID. We have shown that PID excludes PTL expression
from sepal initiation regions. Furthermore, the absence of PID
expression in inter-sepal zones would allow PTL to be expressed
there, hence resulting in suppression of growth in these zones.
PETAL LOSS may influence petal initiation and orientation indirectly
Although loss of PTL function severely affects petal development,
PTL is not expressed in petal primordia until they start to expand at
stage 8, and even then it occurs only in their basal margins
(Fig. 7). Consistent with this,
when PTL is ectopically expressed strongly in the second floral whorl
using the AP3 promoter, petal initiation is frequently abolished, but
overexpression of PTL controlled by its own regulatory sequences does
not disrupt petal initiation. Thus it seems likely that PTL influences petal
development indirectly (assuming it acts cell autonomously). It is true that
PTL function is required for expression of the RBE petal
development gene (Takeda et al.,
2004), but this positive regulation may occur earlier (stages 3-4)
in the inter-sepal zone, where their expression patterns overlap.
|
In ptl mutants, disruption of petal orientation within the flower
is also seen (Griffith et al.,
1999). Petal primordia normally initiate when cells in the L2
layer divide outwards (periclinally) rather than laterally (anticlinally)
(Hill and Lord, 1989
). Sector
analysis has suggested that two adjacent cells are initially involved
(Bossinger and Smyth, 1996
).
Presumably, these cells usually lie side by side on a circumference of the
flower primordium, defining the ultimate orientation of the developing petal.
In ptl mutants, however, petals arise in any orientation
(Griffith et al., 1999
). It
may be that additional growth within the petal-initiating zone in ptl
mutants has relieved the constraint that only cells lying adjacent on a
circumference commence petal development.
In addition to adopting an orientation within the flower primordium, the
two faces of developing petals acquire adaxial-abaxial polarity. Petal
orientation, once established, may automatically define the later developing
polarity, or they may be independent processes
(Griffith et al., 1999). It
has been proposed that polarity of lateral organs is defined by a signal
emanating from the center of the shoot or flower meristem
(Eshed et al., 2001
;
McConnell et al., 2001
;
Emery et al., 2003
). This
could ensure that adaxializing genes of the class III HD-ZIP family are
expressed in adaxial regions of newly arising organ primordia. In ptl
mutants, disruption to such a signal is also likely to be indirect because
PTL is not expressed in petal primordia, the targets of the
signal.
PETAL LOSS may regulate the marginal expansion of leaves and floral organs
Strikingly, PTL expression occurs in the margins of most
developing lateral organs (Fig.
7). Of floral organs, only carpels lack such expression. One
appealing hypothesis is that the PTL protein dampens the growth in these
regions of lateral organs, helping sculpt their final shape. Thus, PTL may
moderate extension around the edges of leaves and sepals, ensuring that they
keep pace with expansion of more centrally located tissue. Also, constriction
in the region of the petiole of leaves, the claw of petals, and the
filament/anther boundary of stamens may be the consequence of growth
suppression. Flower primordia, too, may be constrained from lateral expansion
by early PTL expression in their flanks.
Even so, loss of PTL function does not seem to be associated with any
obvious phenotypic consequences in these regions. Other genes with the same
growth suppression function may still be active (see below). The
homeodomain-encoding gene PRESSED FLOWER is expressed specifically in
lateral regions of primordia of all of these organ types, and in newly arising
flower primordia (Matsumoto and Okada,
2001). However, it apparently promotes growth in lateral regions,
and it will be interesting to assess whether it competes or otherwise
interacts with PTL.
PETAL LOSS is the first trihelix transcription factor known to play a role in morphogenesis
Twenty-eight trihelix genes have been identified in the
Arabidopsis genome (Riechmann et
al., 2000), but only three others have been characterized in
detail to date. All were identified as encoding proteins that bind
specifically to promoter elements required for light responsiveness. The GT-2
protein of rice binds to GT boxes in the promoter of the phytochrome
A gene and is associated with its dark induction
(Dehesh et al., 1990
). A
closely related protein, DF1 from pea, also binds to regulatory elements
necessary for dark induction (Nagano et
al., 2001
). Contrastingly, GT-1a from tobacco binds promoter
elements associated with induction by light
(Gilmartin et al., 1992
;
Perisic and Lam, 1992
).
In addition to PTL, six of the 28 trihelix genes in
Arabidopsis have (or probably once had) duplicated trihelix domains.
These include AtGT-2 (Kuhn et al.,
1993) and AtDF1 (Nagano et
al., 2001
), but not AtGT-1a
(Hiratsuka et al., 1994
). Even
so, neither AtGT-2 nor AtDF1 is closely similar to PTL (60-65% identical in
amino acid sequence in the N-terminal trihelix, and 42-45% in the C-terminal
trihelix). This divergence is apparently reflected by divergence in the
targets of regulation, as PTL does not play any obvious role in light
response.
Expression of PTL in early flower primordia and the inter-sepal
zone is controlled intragenically, probably by sequences within the intron.
Other genes are known that are regulated by intronic elements (e.g.
Sieburth and Meyerowitz,
1997). Interestingly, an intron occurs at this position only in
those 7 members of the trihelix family that arose following duplication of the
trihelix. The closest relative of PTL, At3g10000 (94% identical in
the N-terminal trihelix), shares some tracts of similar sequence within its
intron. It will be of interest to see whether it shares functions with
PTL in defining the inter-sepal zone. Also, it may be that this and
other very close relatives of PTL act redundantly in lateral organ
margins where loss of PTL function is apparently without effect.
In conclusion, this study has revealed that the PETAL LOSS gene helps to control morphogenesis of the perianth. It may repress growth between sepal primordia keeping them separate, and later allow petal developmental signals to be perceived appropriately. This represents a new developmental function for a family of transcription factors previously known only for their role in regulating light response.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Institute of Molecular and Cell Biology, 30 Medical Drive,
Singapore 117609, Republic of Singapore
Present address: Plant Science Center, RIKEN, Yokohama, Kanagawa 230-0045,
Japan
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aida, M., Ishida, T., Fukaki, H., Fujisawa, H. and Tasaka,
M. (1997). Genes involved in organ separation in
Arabidopsis: an analysis of the cup-shaped cotyledon mutant.
Plant Cell 9,841
-857.
Benjamins, R., Quint, A., Weijers, D., Hooykaas, P. J. and Offringa, R. (2001). The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport. Development 128,4057 -4067.[Medline]
Bennett, S. R. M., Alvarez, J., Bossinger, G. and Smyth, D. R. (1995). Morphogenesis in pinoid mutants of Arabidopsis thaliana. Plant J. 8, 505-520.[CrossRef]
Bossinger, G. and Smyth, D. R. (1996).
Initiation patterns of flower and floral organ development in Arabidopsis
thaliana. Development 122,1093
-1102.
Christensen, S. K., Dagenais, N., Chory, J. and Weigel, D. (2000). Regulation of auxin response by the protein kinase PINOID. Cell 100,469 -478.[Medline]
Chuang, C.-F., Running, M. P., Williams, R. W. and Meyerowitz,
E. M. (1999). The PERIANTHIA gene encodes a bZIP
protein involved in the determination of floral organ number in
Arabidopsis thaliana. Genes Dev.
13,334
-344.
Day, C. D., Galgoci, B. F. and Irish, V. F.
(1995). Genetic ablation of petal and stamen primordia to
elucidate cell interactions during floral development.
Development 121,2887
-2895.
Dehesh, K., Bruce, W. B. and Quail, P. H. (1990). A trans-acting factor that binds to a GT-motif in a phytochrome gene promoter. Science 250,1397 -1399.[Medline]
Dehesh, K., Smith, L. G., Tepperman, J. M. and Quail, P. H. (1995). Twin autonomous bipartite nuclear localization signals direct nuclear import of GT-2. Plant J. 8, 25-36.[CrossRef][Medline]
Durfee, T., Roe, J. L., Sessions, R. A., Inouye, C., Serikawa,
K., Feldmann, K. A., Weigel, D. and Zambryski, P. C. (2003).
The F-box-containing protein UFO and AGAMOUS participate in
antagonistic pathways governing early petal development in Arabidopsis.Proc. Natl. Acad. Sci. USA
100,8571
-8576.
Emery, J. F., Floyd, S. K., Alvarez, J., Eshed, Y., Hawker, N. P., Izhaki, A., Baum, S. F. and Bowman, J. L. (2003). Patterning of vasculature and lateral organs by Class III HD-ZIP genes and their miRNAs in Arabidopsis. Curr. Biol. 13,1768 -1774.[CrossRef][Medline]
Eshed, Y., Baum, S. F. and Bowman, J. L. (1999). Distinct mechanisms promote polarity establishment in carpels of Arabidopsis. Cell 99,199 -209.[Medline]
Eshed, Y., Baum, S. F., Perea, J. V. and Bowman, J. L. (2001). Establishment of polarity in lateral organs of plants. Curr. Biol. 11,1251 -1260.[CrossRef][Medline]
Gilmartin, P. M., Memelink, J., Hiratsuka, K., Kay, S. A. and
Chua, N.-H. (1992). Characterization of a gene encoding a DNA
binding protein with specificity for a light-responsive element.
Plant Cell 4,839
-849.
Gleave, A. P. (1992). A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20,1203 -1207.[Medline]
Griffith, M. E., da Silva Conceição, A. and Smyth,
D. R. (1999). PETAL LOSS gene regulates
initiation and orientation of second whorl organs in the Arabidopsis
flower. Development 126,5635
-5644.
Heisler, M. G., Atkinson, A., Bylstra, Y. H., Walsh, R. and
Smyth, D. R. (2001). SPATULA, a gene that controls
development of carpel margin tissues in Arabidopsis, encodes a bHLH
protein. Development
128,1089
-1098.
Hill, J. P. and Lord, E. M. (1989). Floral development in Arabidopsis thaliana: a comparison of the wild type and the pistillata mutant. Can. J. Bot. 67,2922 -2936.
Hiratsuka, K., Wu, X., Fukuzawa, H. and Chua, N.-H.
(1994). Molecular dissection of GT-1 from Arabidopsis.
Plant Cell 6,1805
-1813.
Jack, T., Brockman, L. and Meyerowitz, E. M. (1992). The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell 68,683 -697.[Medline]
Kuhn, R. M., Caspar, T., Dehesh, K. and Quail, P. H. (1993). DNA binding factor GT-2 from Arabidopsis.Plant Mol. Biol. 23,337 -348.[Medline]
Laufs, P., Coen, E., Kronenberger, J., Traas, J. and Doonan,
J. (2003). Separable roles of UFO during floral
development revealed by conditional restoration of gene function.
Development 130,785
-796.
Le Gourrierec, J., Li, Y.-F. and Zhou, D.-X. (1999). Transcriptional activation by Arabidopsis GT-1 may be through interaction with TFIIA-TBP-TATA complex. Plant J. 18,663 -668.[CrossRef][Medline]
Mandel, M. A., Gustafson-Brown, C., Savidge, B. and Yanofsky, M. F. (1992). Molecular characterization of the Arabidopsis floral homeotic gene APETALA1.Nature 360,273 -277.[CrossRef][Medline]
Matsumoto, N. and Okada, K. (2001). A homeobox
gene, PRESSED FLOWER, regulates lateral axis-dependent
development of Arabidopsis flowers. Genes
Dev. 15,3355
-3364.
McConnell, J. R., Emery, J., Eshed, Y., Bao, N., Bowman, J. and Barton, M. K. (2001). Role of PHABULOSA and PHAVOLUTA in determining radial patterning in shoots. Nature 411,709 -713.[CrossRef][Medline]
Moore, I., Gälweiler, L., Grosskopf, D., Schell, J. and
Palme, K. (1998). A transcription activation system for
regulated gene expression in transgenic plants. Proc. Natl. Acad.
Sci. USA 95,376
-381.
Nagano, Y. (2000). Several features of the
GT-factor trihelix domain resemble those of the Myb DNA-binding domain.
Plant Physiol. 124,491
-494.
Nagano, Y., Inaba, T., Furuhashi, H. and Sasaki, Y.
(2001). Trihelix DNA-binding protein with specificities for two
distinct cis-elements: both important for light down-regulated and
dark-inducible gene expression in higher plants. J. Biol.
Chem. 276,22238
-22243.
Perisic, O. and Lam, E. (1992). A tobacco DNA
binding protein that interacts with a light-responsive box II element.
Plant Cell 4,831
-838.
Reinhardt, D., Pesce, E.-R., Stieger, P., Mandel, T., Baltensperger, K., Bennett, M., Traas, J., Friml, J. and Kuhlemeier, C. (2003). Regulation of phyllotaxis by polar auxin transport. Nature 426,255 -260.[CrossRef][Medline]
Riechmann, J. L., Heard, J., Martin, G., Reuber, L., Jiang,
C.-Z., Keddie, J., Adam, L., Pineda, O., Ratcliffe, O. J., Samaha, R. R. et
al. (2000). Arabidopsis transcription factors: genome-wide
comparative analysis among eukaryotes. Science
290,2105
-2110.
Sakai, H., Medrano, L. J. and Meyerowitz, E. M. (1995). Role of SUPERMAN in maintaining Arabidopsis floral whorl boundaries. Nature 378,199 -203.[CrossRef][Medline]
Sieburth, L. E. and Meyerowitz, E. M. (1997).
Molecular dissection of the AGAMOUS control region shows that
cis elements for spatial regulation are located intragenically.
Plant Cell 9,355
-365.
Smyth, D. R., Bowman, J. L. and Meyerowitz, E. M.
(1990). Early flower development in Arabidopsis. Plant
Cell 2,755
-767.
Takeda, S., Matsumoto, N. and Okada, K. (2004).
RABBIT EARS, encoding a SUPERMAN-like zinc finger protein, regulates
petal development in Arabidopsis thaliana. Development
131,425
-434.
Weigel, D. and Glazebrook, J. (2002). Arabidopsis: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Zhou, D.-X. (1999). Regulatory mechanism of plant gene transcription by GT-elements and GT-factors. Trends Plant Sci. 4,210 -214.[CrossRef][Medline]
Zhou, Y., Wang, H., Gilmer, S., Whitwill, S., Keller, W. and Fowke, L. C. (2002). Control of petal and pollen development by the plant cyclin-dependent kinase inhibitor ICK1 in transgenic Brassica plants. Planta 215,248 -257.[CrossRef][Medline]