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First published online April 24, 2009
doi: 10.1242/10.1242/dev.035436
Research Report |
1 Division of Biology 156-29, California Institute of Technology, Pasadena,
California 91125, USA.
2 Laboratoire RDP, Ecole Normale Supérieure de Lyon, 46 allée
d'Italie, 69007 Lyon, France.
3 Temasek Life Sciences Laboratory, National University of Singapore, Singapore
117604, Singapore.
4 Smurfit Institute of Genetics, Trinity College Dublin, College Green, Dublin
2, Ireland.
* Author for correspondence (e-mail: pradeep.das{at}ens-lyon.fr)
* Author for correspondence (e-mail: meyerow{at}its.caltech.edu)
Accepted 16 March 2009
SUMMARY
In Arabidopsis, the population of stem cells present in young flower buds is lost after the production of a fixed number of floral organs. The precisely timed repression of the stem cell identity gene WUSCHEL (WUS) by the floral homeotic protein AGAMOUS (AG) is a key part of this process. In this study, we report on the identification of a novel input into the process of floral stem cell regulation. We use genetics and chromatin immunoprecipitation assays to demonstrate that the bZIP transcription factor PERIANTHIA (PAN) plays a role in regulating stem cell fate by directly controlling AG expression and suggest that this activity is spatially restricted to the centermost region of the AG expression domain. These results suggest that the termination of floral stem cell fate is a multiply redundant process involving loci with unrelated floral patterning functions.
Key words: AGAMOUS, Flower development, Stem cells, Arabidopsis
INTRODUCTION
In Arabidopsis, the indeterminate shoot apical meristem (SAM)
produces organs such as leaves and flowers throughout the life of the plant.
By contrast, the determinate floral meristem (FM), from which flowers are
derived, produces a stereotypical number of floral organs: four sepals, four
petals, six stamens and two carpels. Underlying the different behaviors of
these two meristematic tissues are the different properties of their
respective stem cell populations. In the SAM, as well as in the FM, the
expression of the homeodomain gene WUSCHEL (WUS) in a small
group of cells at the center of the structures, the so-called stem cell
organizing center, is essential for maintaining the pool of stem cells. In the
FM, once the correct numbers of floral organs have formed, WUS is
quickly downregulated and the stem cells lost
(Laux et al., 1996
;
Mayer et al., 1998).
The floral identity regulator LEAFY (LFY)
(Schultz and Haughn, 1991;
Weigel et al., 1992) activates
the expression of the homeotic gene AGAMOUS (AG) in the
center of young flower buds, and the AG gene product then acts to downregulate
WUS, leading to a loss of stem cell activity
(Busch et al., 1999;
Lenhard et al., 2001;
Lohmann et al., 2001;
Parcy et al., 1998). However,
loss of LFY function only leads to a delay in the onset of AG
expression, and not to its absence (Weigel
and Meyerowitz, 1993), suggesting that other factors also play a
role in the early activation of AG. One of these factors was recently
shown to be WUS itself, which directly binds AG regulatory sequences
in combination with LFY (Lohmann et al.,
2001). Flowers mutant for AG display stem cell
maintenance phenotypes, resulting in the formation of flowers within flowers,
and also show homeotic transformations of stamens to petals
(Bowman et al., 1989). It has
been suggested that these are functionally distinct activities of AG
(Mizukami and Ma, 1995;
Sieburth et al., 1995), yet
not much is known about how this is regulated: whether AG is regulated at the
RNA level, for example, via the regulation of AG expression in
specific floral domains, or at the protein level, through interactions between
AG and other spatially restricted molecules. Furthermore, it is unclear how AG
shuts down WUS expression and thus the floral stem cell population
(Laux et al., 1996;
Mayer et al., 1998).
In this study, we report on the identification of a novel input into the process of floral stem cell arrest and suggest that this activity is spatially restricted to the centermost region of the AG expression domain.
MATERIALS AND METHODS
Mutagenesis
pan-3 seeds (10,000-15,000; in the L-er accession) were
treated with a 0.3% (v/v) aqueous solution of ethyl methanesulfonate (Sigma)
in a volume of 15 ml for 10 hours, then washed with water for 8 hours (with
hourly changes) before being resuspended in a 0.15% (v/v) agar solution and
sowed on soil 1 cm apart. Seeds from M1 plants were harvested individually and
20-30 M2 plants per M1 line (
1000) were screened for altered floral
phenotypes, which were reconfirmed in the M3 generation. Ten putative
modifiers were retained after re-screening. To identify the mutation in the
novel lfy allele, the genomic coding region was amplified in two
fragments of 1.3 kb and 1.4 kb by PCR (using Ex Taq, Takara) and sequenced.
The mutation was found to be a nonsense mutation (Q162Stop), similar to all
published null alleles.
Plasmid constructs and sequences
Details of primers available upon request. All PCR amplifications were
carried out using the Phusion high fidelity polymerase (Finnzymes). All
constructs were sequenced. To construct the PAN repressor domain
chimeric fusion, we first annealed complementary oligonucleotides carrying the
enhanced SUPERMAN repressor domain motif flanked by BamHI and
BglII sites, and ligated this to the pGEM-T EZ cloning vector
(Promega), to yield pGEM-SRDX. The PAN cDNA was PCR-amplified,
digested with KpnI and BglII and cloned into the
KpnI and BamHI sites of pGEM-SRDX to yield pPD64.1. The
PAN-RD fragment was extracted with BamHI and
BglII, and cloned into pBJ36
(Gleave, 1992) carrying either
the p35S, pPAN or pAP1(1.7-kb) promoters to yield
pPD66.1, pPD199.2 or pPD143.1 respectively. The PAN promoter was
PCR-amplified from the L-er accession and cloned into pBJ36 using the
SalI and KpnI sites. pAP1(1.7-kb) was a
kind gift of Dr Marty Yanofsky (University of California, San Diego, CA, USA).
The promoter-PAN-RD fragments were then extracted from pBJ36
using NotI and ligated to the plant transformation vector pML BART
(Eshed et al., 2001) yielding
pPD74.1, pPD218.1 or pPD171.20, respectively.
For the ethanol-inducible version of PAN-RD under the control of the PAN promoter, we used the MultiSite Gateway Three-Fragment Vector Construction Kit (Invitrogen) to generate a single plasmid harboring the two components. We PCR-amplified a fragment of the LFY::alcR--alcA::ER-GFP pGreen binary vector (gift of Patrick Laufs; INRA, Versailles, France); the alcR gene harboring a 3' nos terminator, followed by a 35S terminator in an inverted orientation. This fragment was recombined with the pDONR 221 vector to generate pENTR-alcR-2xT. We also modified a destination vector (pGreen 0229; gift of Philip Benfey; Duke University, Durham, NC, USA) by inserting the chimeric promoter alcA immediately after, and oriented towards, the attR3 recombination sequence. To do so, the alcA promoter was PCR-amplified from the LFY::alcR--alcA::ER-GFP pGreen vector, digested with SpeI and HinDIII and ligated to the pGreen 0229 binary vector, to yield the dpGreenBar-alcA binary vector. The PAN promoter was PCR-amplified from Col-0 and recombined with pDONR P4-P1R to yield pPD277. The PAN-RD fragment was amplified from pPD269 and recombined with pDONR P2R-P3 to yield pPD317. Finally, the three pENTR vectors (pPD277, pENTR-alcR-2xT and pPD317) were recombined into dpGreenBar-alcA, to yield the PAN::alcR--alcA::PAN-RD binary vector.
The putative bZIP binding sites in the second AG intron were
identified based on the presence of `ACGT' core sequences
(Jakoby et al., 2002). The six
observed sites are (5'-3'): ACTTATACGTACATGT,
AGTCCCACGTGATTAC, TTGATCACGTCATCAC,
TGTAATACGTATTTGT, TATGGAACGTTGTGAT and
TCCATCACGTTTAAAT.
For the p35S::PAN-VP16 construct, VP16 was
PCR-amplified, digested with BamHI and BglII, and ligated to
pBJ36. The PAN-VP16 fragment was then PCR-amplified and
cloned into the pDONR 221 vector (Invitrogen). The triple gateway system was
then used to generate the final p35S::PAN-VP16 plasmid in pdpGreen-BarT. The
reporter construct used in the particle bombardment assays,
pAGi-3', is published as KB31
(Busch et al., 1999).
Plant lines, transgenics and plant growth conditions
All plants were grown at either 16°C or 22°C with continuous white
light, except the pan-3 and L-er plants shown in
Fig. S6, which were grown at long days (22°C) under a combination of white
and gro-lux light. Photos of flowers were taken using either a Zeiss Stemi SV
11 stereomicroscope fitted with a Zeiss Axiocam or a Leica MZ12
stereomicroscope with a Leica DFC320 camera. Some images were adjusted for
clarity by altering the brightness or contrast but any changes were applied
evenly, across the entirety of the picture, without exception.
In situ hybridizations were performed according to published protocols. The WUS antisense RNA probe corresponds to the entire cDNA. Photos were taken on a Nikon Optiphot-2 equipped with a Zeiss Axiocam.
Transgenic plants were generated using standard floral dipping methods.
Transformant lines were selected on soil for resistance to the herbicide
Basta. To determine the copy number of the PAN-RD transgene,
T2 seeds were plated on petri dishes containing 10 µg/ml ammonium
glufosinate (Basta) and the ratio of resistant to sensitive seedlings
determined (75% resistant seedlings indicates the parent had a single
insertion). The sterile 2xPAN-RD and
1xPAN-RD pan-2 plants were used as
pollen donors to fertilize emasculated wild type flowers, and the F1 seeds
were tested as above.
pAP1(1.7-kb)-driven expression patterns were assayed in inflorescences of ethanol-induced pAP1(1.7-kb)::alcR--alcA::ER-GFP lines treated with the water soluble lipophilic dye FM4-64 and imaged on a Zeiss 510 confocal microscope.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) experiments were performed according
to published protocols (Ito et al.,
1997). Inflorescences from plants mutant for the redundant
APETALA1 and CAULIFLOWER genes, and expressing the
dexamethasone-inducible 35S::APETALA-GR transgene
(p35S::AP1-GR ap1-1 cal-1), were used.
Plants were induced as described (Wellmer
et al., 2006) and tissue were collected 5-7 days later for a
synchronized population of flowers. Inflorescences were ground in liquid
nitrogen, resuspended in buffer M1 (10 mM phosphate buffer 0.1 M NaCl, 10 mM
β-mercaptoethanol, 1 M hexylene glycol), fixed with 1% formaldehyde for
10 minutes, washed in buffers M2 [buffer M1 containing 10 mM MgCl2,
0.5% (v/v) Triton X-100] and M3 (10 mM phosphate buffer, 0.1 M NaCl, 10 mM
β-mercaptoethanol) and centrifuged to collect nuclei. Chromatin was
isolated by resuspending the pellet in lysis buffer [1% SDS (w/v), 10 mM EDTA,
50 mM Tris-HCl pH 8.1] containing protease inhibitors (1 µg/ml Leupeptin,
15 µg/ml Aprotinin), incubating on ice for 10 minutes before adding ChIP
dilution buffer (Upstate) supplemented with protease inhibitors. This mixture
was sonicated and then pre-cleared with salmon sperm DNA-treated Protein A
beads (Upstate) at 4°C for 1-3 hours. The solution was centrifuged, an
aliquot of the supernatant kept for use as control for total input DNA (I) and
the rest was incubated overnight with anti-PAN antiserum
(Chuang et al., 1999) that had
been pre-cleared at 4°C for 2 days against leaves and inflorescences from
pan mutant plants. Chromatin-antibody complexes were captured by
incubating with a Protein A slurry at 4°C for 1 hour, centrifuging to
remove unbound chromatin and eluting off the beads with elution buffer [1% SDS
(w/v), 0.1 M NaHCO3]. Histone-DNA crosslinks were reversed with 0.2
M NaCl at 65°C for 4 hours, RNA and protein were degraded with 40 µg/ml
RNase and 40 µg/ml Proteinase K, respectively, and DNA was purified on a
standard PCR purification column (Qiagen). This purified, antibody-bound DNA
(B) was used to determine enrichment using quantitative real-time PCR on an
ABI 7900HT system (Applied Biosystems) with a MUTATOR-LIKE
(MU) locus (At4g03870) serving as control. Relative enrichment levels
were calculated by determining the ratio of the `mean quantity' (calculated by
the ABI software) of antibody-bound DNA (B) to total input DNA (I) for the
control primers (BCTRL/ICTRL) as well as for each
experimental primer pair (BEXP/IEXP), and then
normalizing the ratio of the experimental value to the control value
[(BEXP/IEXP)/(BCTRL/ICTRL)].
Particle bombardments
Three milligrams of one micron gold microcarrier particles were coated with
2.5 µg of the p3'AGi reporter construct alone (for
the control) or with an additional 2.5 µg of the
p35S::PAN-VP16 (for the co-bombardments). DNA was premixed
prior to each coating. Equal aliquots of the coated particles were then placed
onto macrocarriers and bombarded onto onion epidermal cells using a
PDS-1000/He Biolistic Particle Delivery System (Bio-Rad) and 1100-psi rupture
discs. The onion cells were incubated at 25°C for 2-3 days and then
visualized for GUS staining using standard protocols.
RESULTS AND DISCUSSION
A genetic screen uncovers a combined role for LEAFY and PERIANTHIA in floral stem cell regulation
In a mutagenesis experiment designed to identify modifiers of the floral
organ number regulator PERIANTHIA (PAN)
(Chuang et al., 1999;
Running and Meyerowitz, 1996),
we isolated a new lfy allele (see Materials and methods), which we
named lfy-31. When compared with wild-type flowers
(Fig. 1A), lfy single
mutant flowers (of either lfy-31 or the well-described
lfy-6 allele) bear organs that resemble leaf-like or
sepal-like structures instead of sepals, petals or stamens; and sepalloid
organs with stigmatic papillae instead of carpels
(Fig. 1B,C). However, flowers
of pan-3 mutants, which we used in the mutagenesis
experiment, show no defects in floral identity, but rather bear increased
numbers of sepals and petals, and reduced numbers of stamens
(Fig. 1D). Flowers of the
pan-3 lfy-31 double mutant line bear several
notable differences from flowers of either single mutant. First, the overall
number of primary organs is slightly increased with respect to lfy
flowers (Table 1). Second,
whereas the carpelloid structures of lfy mutant flowers are fully or
partially fused (Fig. 1B,C),
those of pan lfy flowers remain unfused
(Fig. 1E,F). Third, ovule-like
structures are often visible within carpels of lfy mutants
(Fig. 1G) but only very rarely
in the pan lfy double mutant (Fig.
1F). Finally, whereas all lfy flowers produce a
determinate number of organs (Weigel et
al., 1992), 89% (n=38) of pan lfy flowers are
indeterminate, such that ectopic floral structures continue to develop
interior to the fourth whorl organs (Table
1; Fig. 1F). As a
further test of this interaction, we generated lines doubly mutant for
pan-3 and the weak lfy-5 allele
(Weigel et al., 1992), and
observed that these flowers also bear unfused carpels and are indeterminate
(see Fig. S1 in the supplementary material). Thus, in plants carrying
mutations in both LFY and PAN, there is an apparent loss of
floral determinacy that is not observed in the single mutants alone.
|
|
). As in the case of wild-type flowers
(Mayer et al., 1998),
WUS mRNA is clearly detectable in the center of very early flower
buds of pan-3 and lfy-6 single mutants, as
well as in pan-3 lfy-31 double mutants
(Fig. 1H-J). In the wild type,
WUS mRNA becomes undetectable by approximately stage 7, when the
carpel primordia first appear (Mayer et
al., 1998). Similarly, in pan-3 and
lfy-6 single mutant flowers, WUS expression is
absent in later-stage flowers (Fig.
1K,L; see Figs S2 and S3 in the supplementary material). By
contrast, in older pan lfy double mutant flowers, WUS
expression is clearly detectable within the broad expanse of tissue at the
center of the flower from where the organs of the interior floral structures
will arise (Fig. 1M; see Fig.
S4 in the supplementary material). These results indicate that the
indeterminacy phenotypes of pan lfy flowers are associated with the
persistence of the stem cell pool, as revealed by the continued expression of
WUS in the stem cell organizing center.
The role of LFY in the center of the floral meristem, via the activation of
AG expression, has been well studied. In addition, three lines of
evidence suggest that PAN might also be active in this region. First, when
certain alleles of pan (such as pan-3) are grown
under specific culture conditions (see Materials and methods), some flowers
(13%, n=84) show slight indeterminacy (see Fig. S5 in the
supplementary material). Second, flowers from plants doubly mutant for
pan and crabs claw (the carpel patterning gene) are
indeterminate, with a reiteration of carpel structures in internal whorls (see
Fig. S6C-F in the supplementary material; Y. Eshed and J. Bowman, personal
communication). Third, pan mutations restore fourth-whorl carpels to
flowers of the superman-1 (sup-1) single
mutant that normally either lack carpels or have staminoid carpels, also
suggesting a role in this domain (see Fig. S6G-I in the supplementary
material) (Running and Meyerowitz,
1996). Because these data reveal a function for PAN in the
presumptive fourth whorl, we hypothesized that the determinacy defects
apparent in lfy pan double mutant flowers were due to a hidden role
for PAN in regulating the floral stem cell population, which lies within the
fourth whorl.
A dominant-negative pan allele induces floral indeterminacy by suppressing AG expression
One explanation for the absence of floral indeterminacy phenotypes in most
pan mutant flowers is that this activity of PAN might be masked by
functional redundancy with other factors. To overcome such redundancy, we
generated a constitutively repressing form of PAN by fusing it to the SUPERMAN
repressor domain motif (Hiratsu et al.,
2003). Such constructions have been used to study bZIP factors in
several model systems (Fukazawa et al.,
2000; Rieping et al.,
1994) and, in Arabidopsis, SUPERMAN repressor domain (RD)
fusions of several transcription factors have been shown to phenocopy their
corresponding loss-of-function mutants
(Baudry et al., 2006;
Hiratsu et al., 2003;
Xu et al., 2006). The
expression of such a PAN fusion, either ubiquitously or from the
endogenous promoter, yielded plants with severe growth defects, thereby
masking any floral phenotypes (data not shown). In order to assay its effects
on floral patterning, we thus used the flower-specific APETALA1
(AP1) promoter (Hempel et al.,
1997). A 1.7 kb upstream fragment of the AP1 locus drives
expression throughout the early flower; in addition, and at variance with the
normal AP1 expression pattern, it remains active in the entire
flower, presumably due to the absence of additional regulatory elements
(Fig. 2A; see Fig. S7 in the
supplementary material; M. Yanofsky, personal communication).
|
;
Maizel and Weigel, 2004) to
drive PAN-RD expression under the endogenous PAN
promoter. In the absence of induction, pan-2 plants bearing
this construct show no fourth-whorl phenotypes (see Fig. S8A in the
supplementary material). However, after induction with ethanol, we observe
flowers bearing unfused gynoecia and ectopic internal carpel structures (see
Fig. S8B in the supplementary material). Thus, expression of a PAN-repressor
domain fusion protein in the flower leads to the loss of floral determinacy, a
phenotype observed at low frequency in pan mutant plants grown in
specific culture conditions (see above). A caveat to the use of dominant-negative alleles is that they may act as neomorphs, altering the expression of ectopic, rather than genuine, downstream targets of the protein under study. We decided to study this by varying the ratio of chimeric to wild-type protein. If PAN-RD behaves as a true dominant-negative allele, increasing doses of it should yield increasingly stronger phenotypes, whereas increasing doses of unmodified PAN should attenuate the phenotypes. We first used pollen from the PAN-RD/+; pan/pan flowers described above in a cross to wild type and examined the phenotypes of F1 plants selected for the presence of the transgene. We found that these flowers (PAN-RD/+; pan/+) had phenotypes similar to the pan mutant (compare Fig. 2E with 2B). Thus the same PAN-RD insertion that confers strong indeterminacy phenotypes on pan flowers does not do so on pan/+ heterozygotes (instead yielding only the more sensitive organ number defect), showing that PAN-RD expression does not ectopically induce floral indeterminacy. Next we examined primary transformants in wild-type plants, thus with two additional wild-type copies of PAN (Fig. 2F). Approximately 20% (n=60) of these plants (genotypically PAN-RD/+; PAN/PAN) phenocopied the pan mutant (Fig. 2G; 4.3±0.5 sepals, 4.9±0.3 petals and 4.7±0.5 stamens; n=20), whereas the rest showed no discernable phenotypes. We then examined the progeny of these plants, to determine the phenotypes of plants harboring two copies of the transgene (see Materials and methods). These flowers (PAN-RD/PAN-RD; PAN/PAN) displayed strong phenotypes, including extra carpels and floral indeterminacy (Fig. 2H), and closely resembled PAN-RD/+; pan/pan flowers (Fig. 2C,D). Taken together, these data show that the effects of the PAN-RD fusion protein are modified by wild-type PAN in a dosage sensitive manner. This suggests that PAN-RD and endogenous PAN compete for the same targets and that the PAN-RD phenotypes are due to the repression of genuine PAN targets.
To characterize the molecular basis of the PAN-RD phenotype, we performed in situ hybridizations to determine whether the PAN-RD transgene induced changes in AG expression patterns. We observed that, as in the wild type, AG is expressed uniformly throughout the third and fourth whorls of stage 5-6 pan-2 flowers (Fig. 2I). However, in similarly staged PAN-RD/+; pan/pan flowers, AG expression is patchy, with the central region of the floral meristem showing reduced expression (Fig. 2J,K). Since pAP1(1.7-kb) drives expression throughout the central dome of the flower during these stages, these results indicate that the PAN-RD chimera might exert an unequal influence on different cells within the AG-expressing region.
Thus our results show that expression of a PAN-RD fusion protein in the flower is sufficient to mimic pan loss-of-function phenotypes and to produce floral indeterminacy. Since the indeterminacy phenotype occurs more stably in PAN-RD plants than in pan simple mutants, this role possibly requires other spatially or temporally restricted factors. Taken together with the phenotypes of the double mutant flowers described above, this suggests that PAN plays a role in the development of the fourth whorl, specifically in the proper regulation of the floral stem cell population, and that this role is achieved through the regulation of AG. Furthermore, as the effects of PAN-RD were similar to the loss-of-function phenotypes of pan mutant alleles, the role of PAN in floral determinacy is likely to be that of an activator of a gene involved in the process.
PAN binds AG regulatory sequences in vivo
We next sought to determine the precise mechanism by which PAN affects
floral stem cell fate. As discussed above, AG is a key regulator of this
process, by itself acting to repress WUS expression. Because
AG expression is perturbed in PAN-RD flowers and
because PAN is a predicted transcriptional activator, we reasoned that its
role might be to positively regulate AG expression. To test whether
PAN directly associates with the AG promoter, we used chromatin
immunoprecipitation (ChIP) assays, which examine the in vivo binding of
transcription factors to DNA. We maximized the sensitivity of our assays by
using a synchronized population of flowers at stages 5-7
(Wellmer et al., 2006) (see
Materials and methods), as the stem cell organizing activity is known to be
terminated during this time. In addition, we used a characterized PAN-specific
polyclonal antibody that, when used in immunohistochemical analyses, detects
protein signals closely resembling PAN mRNA expression patterns, but
showing no signal in mutant flowers (Chuang
et al., 1999). We further pre-cleared this antibody against tissue
from pan-2 plants prior to immunoprecipitating intact
protein-DNA complexes. We then performed quantitative real-time RT-PCR to
assay for the enrichment of sequences within the 3 kb second intron of the
AG locus with respect to input DNA, where important cis regulatory
sequences are located (Fig. 3A)
(Busch et al., 1999;
Deyholos and Sieburth, 2000;
Hong et al., 2003;
Lohmann et al., 2001;
Parcy et al., 1998;
Sieburth and Meyerowitz,
1997). We observed that five amplicons out of a total of eight
tested within this region are significantly enriched
(Fig. 3B; amplicons
B=9.2-fold±2.0, C=9.0±0.6, F=6.6±0.7, G=4.9±0.4
and H=40.5±9.2) when normalized relative to internal genomic controls.
These data demonstrate that PAN, either alone or in a complex, binds
AG regulatory sequences in vivo.
|
). Of these, amplicon C contains binding sites for LFY and WUS
(Lohmann et al., 2001;
Parcy et al., 1998), whereas
predicted bZIP core binding sites (Jakoby
et al., 2002) are located within or close to two others (F and H).
Hong et al. (Hong et al.,
2003) have shown that an AG reporter containing a
deletion within amplicon H loses expression at later floral stages, suggesting
that it plays a role in the maintenance of AG expression. In an
accompanying article (Maier et al.,
2009), the authors use one-hybrid assays to show that amplicon F
contains PAN binding sites. Furthermore, they show that mutations in this bZIP
motif disrupt binding in vitro and eliminate in vivo expression in the context
of an AG reporter. Given the very high enrichment of amplicon H in
our ChIP assays, we were interested in determining whether this region might
also contain PAN binding sites. To this end, we made use of a published
3' AG reporter construct that reproduces the normal AG
expression pattern in vivo (Busch et al.,
1999). In this reporter, an 800 bp 3' pAG fragment
(Fig. 3A) drives expression of
the uidA gene, which encodes the β-glucuronidase (GUS) enzyme
(Busch et al., 1999). We
performed particle co-bombardment experiments in onion epidermal cells with a
putative constitutively activated form of PAN
(p35S::PAN-VP16). Although the reporter alone presents basal
reporter activity in onion cells (44.5±34.6 cells;
Fig. 3C; see Fig. S9A,C in the
supplementary material), perhaps due to the presence of minimal p35S
sequences, 4- to 5-fold greater numbers of cells (157±11.3;
Fig. 3C; see Fig. S9B,D in the
supplementary material) show GUS activity when co-bombarded with
p35S::PAN-VP16. It is thus possible that PAN has two or more
target sites within the AG promoter, to which it might bind with
different affinities or different partners.
Flowers mutant for AG display stem cell overproliferation
phenotypes and, in addition, also show homeotic transformations of stamens to
petals (Bowman et al., 1989).
Since PAN-RD disrupts floral stem cell regulation without
inducing the homeotic transformations associated with the loss of AG
function, we asked whether PAN might function as a general regulator of
AG, or only to modify its activity in the fourth whorl. To this end,
we used the weak ag-4 allele, which produces flowers with
reduced numbers of stamens and with fourth whorl organs replaced by another
flower (Fig. 4A)
(Sieburth et al., 1995).
Mutations in several loci, including HUA1 and HUA2, REBELOTE
(RBL) or ULTRAPETALA1 (ULT1) enhance the
ag-4 allele by fully or partially converting stamens to
petals (Chen and Meyerowitz,
1999; Fletcher,
2001; Prunet et al.,
2008). We reasoned that if PAN plays a general role in regulating
AG expression, a pan allele might similarly enhance the weak
ag phenotype. Conversely, if PAN participates primarily in the floral
determinacy aspects of AG, the double mutant might not be
significantly enhanced. In fact, we observe that pan-2
ag-4 double mutant flowers
(Fig. 4B) differ from
ag-4 single mutants only in that they present an additional
pan-like phenotype: extra perianth organs (4.8±0.5 sepals and
4.8±0.5 petals compared with four each in ag-4;
n=20 for both genotypes), as is the case for double mutants of
pan and the strong ag-1 allele
(Running and Meyerowitz,
1996). The third-whorl stamens of pan-2
ag-4 flowers appear morphologically normal, although they are
reduced in number (5.5±0.5 compared with 5.8±0.4 in
ag-4). This suggests either that the stamen identity
functions of the mutant protein encoded by the ag-4 allele
are robust and are not perturbed by the absence of PAN, or that PAN regulates
AG expression only in the fourth whorl.
|
) have recently proposed the existence of a distinct subdomain
within the floral fourth whorl, in which a decrease in AG expression
is sufficient to disrupt the specification of floral determinacy. Two
observations indicate that the effects of PAN on the AG promoter
might be spatially restricted, perhaps to this subdomain. First, in
PAN-RD flowers, there is a marked reduction in the
accumulation of AG transcript at the very center of the dome of the
floral meristem. Second, unlike many other AG interactors
(Chen and Meyerowitz, 1999;
Fletcher, 2001;
Prunet et al., 2008),
pan mutations have no effect on the third whorl phenotypes of a weak
ag allele. A restricted effect of PAN on AG expression might
explain why mutations in PAN attenuate the fourth whorl phenotype of
sup mutants (Running and
Meyerowitz, 1996), which produce reduced or masculinized carpels
(see Fig. S6G,H in the supplementary material)
(Bowman et al., 1992). One
model is that in the center of the fourth whorl of pan sup double
mutant flowers (see Fig. S6I in the supplementary material), even the mild
reduction in AG expression caused by the absence of PAN could lead to
increased or prolonged WUS expression, and thus to an increase in the
size of this region. Since the stamen identity genes AP3 and
PI are excluded from the very center of sup flowers
(Bowman et al., 1992;
Goto and Meyerowitz, 1994),
the corresponding, but enlarged, region in pan sup flowers could now
become specified into a functional carpel.
Like PAN, the broadly expressed APETALA2 (AP2)
gene, the absence of which causes only specific floral phenotypes (a change in
sepal and petal identities), was recently shown, through the characterization
of a semi-dominant allele, to play a role in the control of stem cells in the
shoot (Würschum et al.,
2006). Thus both PAN and AP2 have primary roles in specific
aspects of floral patterning, but also have masked functions in stem cell
regulation that are likely to require interactions with other domain- and/or
stage-specific factors. Identifying these interactors through genetic screens
or other methods could prove invaluable in gaining a full understanding of the
complex regulatory mechanisms that control stem cell fate.
As the main function of AG in floral determinacy appears to be to downregulate WUS expression in a narrow temporal window, AG expression must be quickly upregulated in order to ensure the complete arrest of stem cell fate, and thereby ensure proper floral patterning. We suggest that plants have evolved a complex, multiply redundant system to ensure the proper regulation of AG, and furthermore, that many of the factors involved in the regulation of floral stem cells probably also perform unrelated patterning functions.
Footnotes
Supplementary material available online at http://dev.biologists.org/cgi/content/full/136/10/1605/DC1
We thank J. Lohmann for sharing unpublished results; Arnavaz Garda, Alexis Lacroix, Claudia Bardoux and Hervé Leyral for technical assistance; Ioan Negrutiu, Christophe Trehin, Nathanaël Prunet and Olivier Hamant for useful discussions and comments on the manuscript; Ioan Negrutiu for sharing unpublished results and seeds; and John Bowman and Marty Yanofsky for communicating unpublished results. This work was supported by a Caltech Division of Biology postdoctoral fellowship (to P.D.), a Marie Curie Incoming International Fellowship IIF-022002 (to P.D.), the EU Marie Curie SY-STEM Network (to J.T.) and a NSF Grant IOS-0544915 (to E.M.M.).
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