Direct regulation of the floral homeotic ... - Wiley Online Library

The Plant Journal (2006) 46, 593–600

doi: 10.1111/j.1365-313X.2006.02720.x

Direct regulation of the floral homeotic APETALA1 gene by APETALA3 and PISTILLATA in Arabidopsis Jens F. Sundstro¨m1,2,*, Naomi Nakayama1, Kristina Glimelius2 and Vivian F. Irish1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520-8104, USA, and 2 Department of Plant Biology and Forest Genetics, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden

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Received 19 September 2005; revised 16 January 2006; accepted 23 January 2006. *For correspondence (fax þ46 18 673279; e-mail jens.sundstrom@vbsg.slu.se).

Summary The floral homeotic gene APETALA1 (AP1) specifies floral meristem identity and sepal and petal identity in Arabidopsis. Consistent with its multiple roles during floral development, AP1 is initially expressed throughout the floral meristem, and later its expression becomes restricted to sepal and petal primordia. Using chromatin immunoprecipitation, we show that the floral homeotic PISTILLATA (PI) protein, required for petal and stamen development, has the ability to bind directly to the promoter region of AP1. In support of the hypothesis that PI, and its interacting partner APETALA3 (AP3), regulates the transcription of AP1, we show that AP1 transcript levels are elevated in strong ap3-3 mutant plants. Kinetic studies, using transgenic Arabidopsis plants in which both AP3 and PI are under post-translational control, show that AP1 transcript levels are downregulated within 2 h of AP3/PI activation. This implies that the reduction in AP1 transcripts is an early event in the cascade following AP3/PI induction and provides independent support for the hypothesis that AP1 is a direct target of the AP3/PI heterodimer. Together these results suggest a model whereby AP3/PI directly acts, in combination with other factors, to restrict the expression of AP1 during early stages of floral development. Keywords: MADS-box genes, flower development, transcription factors.

Introduction The transition from vegetative growth to reproductive development in Arabidopsis thaliana is, in part, regulated by the activity of the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1). When the activity of both of these genes is compromised, inflorescence shoots develop in place of flowers (Huala and Sussex, 1992; Weigel et al., 1992). In the strong lfy-6 mutant, flowers are replaced by leaves and second-order shoots (Weigel et al., 1992), whereas in the strong ap1-1 mutant, flowers have partial shoot characters (Bowman et al., 1993; Irish and Sussex, 1990). Genetic and molecular evidence suggests that AP1 is positively regulated by LFY, as AP1 expression is delayed and reduced in lfy mutants (Liljegren et al., 1999; Mandel et al., 1992; Ratcliffe et al., 1999; Ruiz-Garcia et al., 1997) and AP1 expression can be directly activated in transgenic plants harbouring an inducible form of LFY (Wagner et al., 1999). The expression of AP1 in the floral meristem is also dependent on flowering time genes such as FT and FD (Abe et al., 2005; Huang et al., 2005; Wigge et al., 2005). ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd

Apart from being a floral meristem identity gene, AP1 is also involved in directing floral organ development, as ap1 mutants show homeotic conversions of sepals into bracts and loss or reduction of petals (Bowman et al., 1991, 1993; Irish and Sussex, 1990). In agreement with its dual function, AP1 mRNA accumulates uniformly throughout the floral meristem during the first stages of floral development, but becomes restricted to the outer two whorls as sepal and petal development initiates (Mandel et al., 1992). According to mRNA in situ expression analysis, this spatial restriction is mediated by the floral homeotic AGAMOUS (AG) protein, which negatively regulates the expression of AP1 in the third and fourth whorls (Gustafson-Brown et al., 1994). The AP1 expression domain is not noticeably affected in plants mutated for the APETALA3 (AP3) gene, which mediates petal and stamen identities (Gustafson-Brown et al., 1994). Whereas AP1 has been shown to directly and indirectly regulate the early expression of AP3 and its interacting partner, PISTILLATA (PI) (Lamb et al., 2002; Ng and Yanofsky, 2001), late AP3 and PI expression depends on their own 593

594 Jens F. Sundstro¨m et al.

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Apart from AP3, only one gene, NAP (NAC-LIKE ACTIVATED BY AP3/PI), has been identified as being directly regulated by the AP3/PI heterodimer (Sablowski and Meyerowitz, 1998). To screen for novel candidate genes regulated by AP3, we used Arabidopsis plants harbouring an inducible form of AP3. These plants contain a transgene consisting of AP3 cDNA fused to the hormone-binding domain of the rat glucocorticoid receptor (GR) under the control of the constitutive cauliflower mosaic virus 35S promoter (Sablowski and Meyerowitz, 1998) and are of the genetic constitution ap3-3; 35S::PI; 35S::AP3–GR (abbreviated as AP3–GR). Hence, both AP3 and PI proteins are present in all four floral whorls of these plants, but AP3 protein is retained in the cytosol if the plants are grown without dexamethasone (DEX). Upon DEX induction, the AP3–GR protein translocates to the nucleus where it can heterodimerize with the PI protein and affect transcription. To identify genes regulated by the activation of AP3 function, flowers of AP3–GR plants were treated for 6 h with DEX or a mock solution (MOCK) and the resulting mRNA populations were compared using the Yale 9.2K Arabidopsis EST microarray (for array design and microarray results see Appendix S1). The experiment was carried out in duplicate, using independently treated biological samples. One of the genes identified as being

To confirm and extend these microarray results, we compared the AP1 transcript levels in inflorescences of strong ap3-3 and pi-1 mutants and plants carrying a 35S::AP3 or 35S::PI transgene with that of the wild type (WT; Figure 1a). Using quantitative real time PCR (qRT-PCR), we showed that AP1 transcript levels were significantly elevated in ap3-3 mutant plants compared with both WT and 35S::AP3 plants (Student’s t-test, P < 0.0001). In addition, we observed a slight reduction in the AP1 transcript levels in plants expressing AP3 constitutively compared with WT (Student’s t-test P < 0.005). Similarly, AP1 transcripts levels were elevated in pi-1 mutants as compared with both WT and 35S::PI plants (Student’s t-test P < 0.0001). We could not detect a significant difference in AP1 expression in 35S::PI plants as compared with WT. These results suggest that AP3 and PI repress AP1 transcription and are in agreement with those reported by Wellmer et al., 2004 for AP1 transcript levels in a microarray screen, in which AP1 transcript levels were elevated in both ap3 and pi mutant plants as compared with the WT. The fact that constitutive expression of either AP3 or PI alone did not result in a dramatic reduction in AP1 transcript levels is not surprising, as AP3 and PI have been shown to act as an obligate heterodimer and are thus both required for biological activity (Krizek and Meyerowitz, 1996). In order to establish whether the elevated transcript levels of AP1 in ap3-3 and pi-1 mutants are due to a shift in the spatial domains of expression, we performed mRNA in situ

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Microarray results imply negative regulation of AP1 by AP3/PI

AP1 transcript level is elevated in ap3-3 and pi-1 mutants

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Results and discussion

negatively regulated by the AP3/PI heterodimer in our microarray screen was AP1.

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gene products, which suggests that the maintenance of AP3 and PI expression is regulated by an autoregulatory feedback loop (Goto and Meyerowitz, 1994). AP3 and PI act as obligate heterodimers and are dependent on each other for nuclear stability (McGonigle et al., 1996). In agreement with this, the AP3 and PI heterodimer has been shown to bind specific sequence elements called CArG-boxes in vitro (Schwarz-Sommer et al., 1992), present in the AP3 promoter (Hill et al., 1998; Tilly et al., 1998). Furthermore, the AP3/PI heterodimer appears to form higher-order protein complexes in combination with other MADS-box proteins that differentially act to specify petal versus stamen identities (Honma and Goto, 2001; Pelaz et al. 2001). Here we examine the possible regulation of AP1 by the homeotic AP3/PI proteins. We show that the loss of AP3 function results in the upregulation of AP1 expression, whereas the induction of AP3 and PI expression results in the loss of AP1 expression. Furthermore, we show that the PI protein directly binds to sequences in the AP1 promoter, suggesting that this regulation is direct. The data presented here, together with previous results, provide evidence to suggest that the genes involved in organ specification regulate the transcriptional activity of each other in a complex network of regulatory feedback loops.

Figure 1. AP1 transcript levels are elevated in the strong ap3-3 and pi-1 mutants. (a) The relative expression levels of AP1 in wild-type (WT), pi-1, 35S::PI, ap3-3 and 35S::AP3 and inflorescences assayed by quantitative real time PCR (qRTPCR). (b) The relative expression levels of AP1 in WT, MOCK-treated AP3–GR plants ()) and dexamethasone-treated AP3–GR plants (þ). Mean values from two technical and two biological repeats are presented. Error bars represent the SD.

ª 2006 The Authors Journal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 593–600

Direct regulation of AP1 by AP3 and PI 595 hybridization experiments. In longitudinal sections of WT inflorescences hybridized with an AP1 antisense probe, no signal was detected in the inflorescence meristem, but throughout stage 1 and 2 flowers (Figure 2a). In later stages, the AP1 signal was restricted to the sepal and petal whorls and absent from the developing stamens and carpels. This pattern was clearly shown in stage 4 flowers, where the young sepals overlie the central floral meristem (Figure 2a). The expression pattern of AP1 in WT flowers was in agreement with previously published data (Mandel et al., 1992). In longitudinal sections of pi-1 inflorescences, AP1 expression could also be detected throughout stage 1 and 2 flowers and, similar to WT, the signal in later stage flowers was restricted to the two outermost whorls (Figure 2b). A similar pattern of AP1 expression was also detected in longitudinal sections of 35S::PI plants (Figure 2c), whereas an AP1 sense control probe showed no signal above background in any of the genotypes (Figure 2d). Similarly, the pattern of AP1 expression is not apparently altered in ap3-3 mutants (Gustafson-Brown et al., 1994). These data indicate that the elevated expression levels of AP1 in pi and ap3 mutants as compared with WT flowers is not due to a shift in AP1 expression domains due to differences in floral organ identity, but rather point to the possibility that AP3/PI regulate the absolute levels of AP1 transcripts.

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Figure 2. AP1 is expressed throughout young flowers and in developing first and second whorl organs in both wild-type (WT) and pi-1 flowers. The figure shows light-field micrographs of longitudinal sections of (a) WT L (er), (b) pi-1 and (c) 35S::PI inflorescences hybridized with a AP1-specific antisense probe. The signal appears as dark brown to purple in colour. (d) Longitudinal section of a WT L (er) inflorescence hybridized with an AP1 sense control probe. Size bar ¼ 100 lm. The numbers indicate the flowers of different stages, according to Smyth et al., 1990. The arrow points to a petal overlaying the central floral meristem in stage 4 flowers in (a), (b) and (d), and a stage 3 flower in (c). The arrowhead points to the inflorescence meristem in all micrographs. p, petal.

The MOCK-treated AP3–GR plants used in our microarray analysis are phenotypically similar to strong ap3 mutants. Accordingly, AP1 expression levels assayed by qRT-PCR (Figure 1b) were elevated in MOCK-treated AP3– GR plants as compared with WT plants (Student’s t-test P < 0.0001), whereas in DEX-treated AP3–GR plants, AP1 transcript levels were similar to those of WT plants. Because both AP3 and PI are probably present in excess in the DEX-treated AP3–GR plants, these results indicate that additional factors are involved in the modulation of AP1 expression in this context. AP1 is downregulated as an early response to AP3/PI induction To circumvent a possible temporal masking of the regulatory effect by AP3/PI on AP1 expression, we examined the kinetics of AP1 expression using the inducible AP3–GR construct (Figure 3a). AP3–GR plants were treated once, at time zero, with either DEX or MOCK solution. Inflorescences were collected at time zero and after 30 min, 2, 6 and 12 h of initial treatment and the relative expression levels were estimated in the DEX and MOCK samples by qRT-PCR. In these studies, AP1 expression levels did not change during the first 30 min following DEX induction. However, AP1 transcript levels were significantly reduced in DEX-treated plants compared with MOCK-treated plants 2 h after steroid treatment (Student’s t-test P < 0.0001). Furthermore, in the samples collected 6 h after DEX induction, the log2 ratio was close to )1, indicating that the AP1 expression level had been reduced two-fold following AP3/PI induction. The low DEX to MOCK ratio persisted for 12 h after induction. These results are in agreement with the results obtained in our microarray screen and provide independent confirmation of those experiments. We have not been able to detect any significant changes in the DEX/MOCK ratio in the 30-min samples for any of our additional candidate genes tested so far (data not shown), which may reflect a lag time before the DEX has entered the cells. Provided that this assumption is valid, the reduction in AP1 transcript levels already apparent in the 2-h sample indicates that the reduction in AP1 transcripts is an early event in the cascade following AP3/PI induction. In addition to AP1, the relative expression levels of two closely related MADS-box genes, CAULIFLOWER (CAL) (Kempin et al., 1995) and FRUITFULL (FUL) (Mandel and Yanofsky, 1995), were estimated in the DEX- and MOCKtreated plants. The relative expression levels of CAL did not change in response to the DEX induction, shown as a log2 ratio close to zero at all time points (Figure 3b). Similarly, the FUL log2 ratios of DEX- and MOCK-treated samples were close to zero in the 30 min and 2 h samples. In the 6 and 12 h samples, the FUL log2 ratio approached )0.4 (Student’s t-test P < 0.05) (Figure 3c).


596 Jens F. Sundstro¨m et al. AP1

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(Figure S1), similar to those previously reported for other 35S::PI transgenes (Krizek and Meyerowitz, 1996), demonstrating that the PI–HA fusion protein is functional and can confer petaloid identity. A monoclonal antibody directed against the HA epitope of the PI–HA fusion protein was used in a ChIP experiment (Figure 4a). Nuclear extracts derived from 35S::PI–HA inflorescences (including floral buds up to stage 12, staged according to Smyth et al., 1990) were sonicated to obtain DNA fragments ranging from 250 to 1000 bp (Figure 4b). After immunoprecipitation, we estimated the enrichment of the promoter regions of AP1 relative to a negative control, transposon Mu-2, using qRT-PCR. Two primer pairs were

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Figure 3. AP1 is two-fold downregulated in response to AP3/PI induction. The graphs show the relative transcript levels of (a) AP1, (b) CAL and (c) FUL at different time points after steroid induction of AP3/PI nuclear translocation. mRNA levels were estimated using quantitative real time PCR (qRT-PCR). The values show the log2 ratio between DEX and MOCK-treated samples. The plant material for RNA preparation was collected at time zero, 30 min, 2, 6 and 12 h after steroid induction. The mean values from two technical and two biological repeats are presented. Error bars represent SD.

Chromatin immunoprecipitation (ChIP) experiments suggest a direct regulation of AP1 by AP3/PI In order to examine whether the early reduction in AP1 transcript levels in our kinetic experiments was due to a direct regulation of AP1 by AP3/PI, we produced transgenic Arabidopsis plants harbouring an Influenza hemaglutinin (HA)-tagged version of the PI protein under the control of the constitutive cauliflower mosaic virus 35S promoter. Flowers of these plants develop sepals with petaloid margins

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Figure 4. AP3 and AP1 promoter regions were enriched by chromatin immunoprecipitation (ChIP) using a monoclonal HA antibody. (a) Western blot comparing protein extracts of PI–HA and wild-type (WT) buds probed with the monoclonal HA antibody. The PI–HA plants produced a fusion protein of approximately 30 kDa. (b) Nuclear extracts derived from PI–HA inflorescences were sonicated to yield DNA fragments in the range of 250–1000 bp. (c) The boxed areas represent the 1000 bp upstream region of either AP1 or AP3. The vertical lines indicate the position of theoretical CArG-boxes in the AP1 and AP3 promoter and the horizontal lines mark regions amplified by PCR primers used in the ChIP experiments. The numbers indicate base pairs relative to each start codon (ATG). (d) The enrichment of promoter regions of AP1 and AP3 estimated by quantitative real time PCR (qRT-PCR). Values of bound over input for each gene were normalized against unspecific enrichment of the transposon Mu-2. The results from two independent ChIP experiments are presented. The PCR reaction efficiency was 1.98 for the AP1 and Mu-2 primers and 1.82 for the AP3 primers (P < 0.0005 in all samples). Error bars represent normalized SE.


Direct regulation of AP1 by AP3 and PI 597 used to estimate the relative enrichment of the AP1 promoter; one pair that amplified a 51 bp region located 416 bp upstream of the AP1 start codon, and a second pair that amplified a 109 bp region located 682 bp upstream of the AP1 start codon, covering a theoretical CArG-box present in the AP1 promoter (Figure 4c). Both primer pairs amplified the AP1 promoter at similar levels (data not shown; for primer sequences see Table S4). In two separate ChIP experiments, the promoter region of AP1 was enriched 4.5and 2.8-fold (Figure 4d). The AP3 promoter was used as a positive control; a primer pair that amplified a 52 bp region located 120 bp upstream of the AP3 start codon (Figure 4c) was used in the qRT-PCR reactions and the AP3 promoter fragment was enriched 2.9- and 1.4-fold (Figure 4d). These results show that PI protein binds to target sequences in the AP1 promoter and suggests a direct regulation of AP1 by the AP3/PI heterodimer. AP3/PI does not appear to regulate CAL or FUL transcription in the same manner as AP1 AP1 acts partially redundantly with a closely related gene, CAL, to determine floral meristem identity (Kempin et al., 1995). Although single cal mutants have no apparent phenotype, ap1 cal double mutants show an enhancement of the ap1 mutant phenotype. Additionally, ap1 cal mutants display a massive proliferation of inflorescence-like meristems in positions that would normally be occupied by flowers (Bowman et al., 1993; Kempin et al., 1995). In agreement with this observation, AP1 and CAL have overlapping expression domains in inflorescence meristems and young flowers until stage 3. Subsequently the CAL expression domain is restricted to fourth whorl organs (Kempin et al., 1995). Double ap1 cal mutant plants do eventually produce flowers, due to the activity of the FUL gene (Ferrandiz et al., 2000). FUL is expressed in the inflorescence meristem and subsequently also becomes activated in the central dome of stage 3 flowers, and later has functions during carpel development (Ferrandiz et al., 2000; Mandel and Yanofsky, 1995). Interestingly, mRNA in situ hybridization in ap3 mutants indicates that AP3 negatively regulates FUL expression, as the domain of FUL expression is expanded to whorl three in stage 3 of ap3 mutants (Mandel and Yanofsky, 1995). We have not been able to demonstrate negative regulation of CAL or FUL by AP3/PI, as shown for AP1. CAL and FUL are not expressed in ap3 and pi mutants in the same coordinate manner as AP1 (Wellmer et al., 2004). In our kinetic experiments using the AP3–GR construct, CAL expression levels did not change in response to DEX induction (Figure 3b), nor could we detect a pronounced downregulation of FUL transcript levels (Figure 3c). In addition, we could not detect any enrichment of either the CAL or FUL promoter regions in our ChIP experiments (data not shown). This

suggests that AP3/PI does not regulate the AP1 paralog CAL and that the previous reported negative regulation of FUL by AP3 appears to be indirect and may be due to the fact that third whorl organs in ap3 mutants have carpeloid identity. Constitutive expression of PI causes an increased frequency of ap1-like chimeric shoots in transgenic Arabidopsis plants We have not been able to detect any large reduction in AP1 transcription levels in any of the transgenic plants that constitutively express either AP3 or PI. However, it is possible that ectopic expression of AP3 and PI may cause a reduction in AP1 expression in a small number of cells at a specific interval during development that may be manifested in a subtle ap1-like phenotype. To assess this possibility, we examined the frequency with which we could detect ap1-like phenotypes in plants ectopically expressing PI. Wild-type Arabidopsis plants produce chimeric flowering shoots, at a low frequency, with similarities to ap1 mutant flowers (Hempel and Feldman, 1995). We compared the frequency of such chimeric flowering shoots in our 35S::PI–HA plants with WT plants (Figure 5 and Table 1). In two separate experiments, we identified 0.98% chimeric flowering shoots in WT plants, whereas chimeric flowering shoots occurred in 5.25% of the PI–HA plants (Table 1). This slight increase in chimeric shoot occurrence was statistically significant (Student’s t-test P < 0.05) and in agreement with the hypothesis that AP3/PI regulates AP1 transcript levels. We

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Figure 5. ap1-like chimeric shoots are formed in the position of the first flower in an inflorescence. SEM of (a) normal and (b) ap1-like chimeric shoot inflorescences. The arrowhead points to a normal first flower in a wild-type (WT) inflorescence in (a) and an ap1-like chimeric shoot in (b). Scale bar ¼ 100 lm.

Table 1 The 35S::PI–HA transgene is associated with a slight increase in frequency of ap1-like flowers. Shown are the average frequencies of ap1-like flowers found on primary or secondary shoots of wild-type (WT) L (er), PI–HA, cal-5 and cal-5*PI–HA plants. The average and SD were calculated from two independent experiments. The total numbers of plants screened (n) are also indicated

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0.98 5.25 7.05 21.25

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598 Jens F. Sundstro¨m et al. also examined the frequency of ap1-like chimeric shoots in a cal mutant background, as CAL is partially redundant with AP1, but AP3 and PI do not regulate CAL in a similar manner; this should provide a more sensitive background in which to recover ap1-like phenotypes. Homozygous cal-5 mutants produced chimeric flowering shoots in 7.05% of the plants, whereas plants homozygous for both cal-5 and the 35S::PI– HA transgene displayed 21.25% chimeric flowering shoots. This difference was statistically significant (Student’s t-test P < 0.05). Hence, ectopic expression of the 35::PI–HA transgene leads to an increase in occurrence of ap1-like chimeric shoots and this phenotype is enhanced in cal mutant plants. The results presented here, and those of Wellmer et al. (2004), indicate that AP3 and PI negatively regulate AP1 expression levels. Interestingly, this negative regulation seems to be independent of floral organ identity, as AP1 is expressed in second whorl organs of both WT and pi-1 or ap3-3 mutant flowers. Additional evidence for this was provided by the kinetic study of reduced AP1 expression in AP3–GR plants, as the reduction in AP1 expression occurred early and before any change in organ identity was observed (data not shown). Here we present evidence for the presence of a direct negative feedback loop between individual floral organ identity genes. Positive auto- and cross-regulation of floral organ identity genes has previously been demonstrated for AP3 and PI (Goto and Meyerowitz, 1994; Jack et al., 1994). AP1 has been shown to positively regulate AP3 and PI (Lamb et al., 2002; Ng and Yanofsky, 2001). In addition, AG has been shown to regulate both AP3 and SEP3 transcription (Gomez-Mena et al., 2005). Hence, a growing number of reports have shown that the floral organ identity genes regulate each other in a co-ordinated manner at the transcriptional level. The presence of a negative feedback loop in the co-ordinated regulation of organ identity genes points to the possibility that the transcriptional activity of the organ identity genes is not only regulated by a cascade of positive regulators, but that transcription levels are determined by a balanced network of both positive and negative autoregulatory feedback loops. This idea is supported by the observation that overall AP1 expression levels are approximately equivalent to WT in all the transgenic lines in our qRT-PCR experiments. This in turn points to the possibility that additional factors are involved in the regulation of AP1, together with AP3/PI. One possible candidate for such a factor could be SEP3, which has been postulated to participate in a multimeric protein complex together with AP3/PI and AP1 to direct petal development (Honma and Goto, 2001), and together with AP3/PI and AG to regulate stamen development (Gomez-Mena et al., 2005; Honma and Goto, 2001). Thus, it is possible that the negative regulation of AP1 by AP3/PI defines an additional regulatory circuit by which floral organ identity gene products cross-regulate their expression levels, and it appears that this complex regula-

tory feedback is of importance for the correct establishment of floral organ identity.

Experimental procedures Plant material Arabidopsis thaliana plants were grown on a 12:3:1 mix of vermiculite:soil:sand at 22C under 16 h light/8 h dark conditions. The mutant lines (ap3-3; pi-1; cal-5) and transgenic lines (ap3-3; 35S::PI; 35S::AP3–GR [AP3–GR], 35S::AP3 and 35S::PI–HA) are in the Landsberg erecta background. The PI–HA fusion construct was made by cloning the PI coding region (for primer sequences see Table S1) into pGWB14, a gateway-compatible binary vector designed for 35S promoter-driven expression of HA fusion proteins (kindly provided by T. Nakagawa, Shimane University, Izumo, Japan). Cloning using gateway vectors was carried out using reagents and protocols from Invitrogen (Carlsbad, CA, USA). Transgenic Arabidopsis plants were generated using the floral dip method (Clough and Bent, 1998) and selected on medium containing 30 mg l)1 kanamycin. The AP3–GR line was a gift from Robert W. M. Sablowski (John Innes Centre, Norwich, UK).

Quantitative real-time PCR experiments (qRT-PCR) Inflorescences with flower buds of stages 1–12 were collected from all genotypes examined and snap frozen in liquid nitrogen. In the kinetic studies, DEX or MOCK treatment was performed once and material was collected at the following time points: 0, 0.5, 2, 6 and 12 h. Total RNA was extracted from plant tissues using Trizol (GibcoBRL, Frederick, MD, USA) according to the manufacturer’s instructions. RNA samples were DNAse treated (Fermentas Inc, Hanover, MD, USA) for 45 min at 37C. cDNA was synthesized using Superscript II RNase-Reverse Transcriptase (Invitrogen) according to the manufacturer’s instructions and checked for purity using specific genomic DNA primers. RT-PCR reactions were carried out using an ABI Prism 7000 Sequence Detector (Applied Biosystems, Foster City, CA, USA) in MicroAmp optical 96-well reaction plates with optical covers, according to the manufacturer’s instructions. PCR reactions (final volume 25 ll) contained SYBRGreen, primers and the passive reference dye ROX, in order to normalize fluorescence across the plate. In all experiments, controls without the template were used and at least three replicates for each sample were included. The experiments were repeated at least twice using independent RNA samples. The reaction conditions were: 50C for 2 min, 94C for 10 min followed by 40 cycles of 94C for 15 sec, 60C for 1 min. Primers were designed using Primer Express software (Applied Biosystems) to flank introns, when possible, so genomic DNA contamination would not amplify (for primer sequences see Table S2). Values were normalized against either the relative expression of b-tubulin or actin-2 in each sample, yielding comparable results (data not shown). Relative quantification values and standard deviations were calculated using the standard curve method, according to the manufacturer’s instructions (ABI Prism 7000 Sequence Detection System Users Guide). Statistical analyses (Student’s t-test) were performed using http://graphpad.com/ quickcalcs/ttest1.cfm.

In situ hybridization Templates for in situ probes were generated by an initial PCR amplification of a gene-specific region of the AP1 transcript, using


Direct regulation of AP1 by AP3 and PI 599 the corresponding full-length cDNA as the template. This was followed by a second round of PCR amplification using the first PCR product as the template, and primers containing T7 RNA polymerase-binding sites in either the sense or antisense direction (for primer sequences see Table S3). Digoxygenin-labelled UTP (Boehringer Mannheim, Indianapolis, IN, USA) probes were transcribed using T7 RNA polymerases. The tissue was fixed in 4% paraformaldehyde (Sigma, St Louis, MO, USA) and embedded in Paraplast X-tra (Monoject Scientific, St Louis, MO, USA), and 8 lm sections were affixed to Probe-on Plus slides at 42C (Fisher Scientific, Pittsburgh, PA, USA). In situ pre-hybridization, hybridization and detection were performed as described previously (Carr and Irish, 1997).

the SEM studies, and Drs Eva Sundberg and Matti Leino for critical reading of the manuscript. Dr Elliot Meyerowitz is acknowledged for hosting JFS during a short-term visit. This work was supported in part by grants from the US National Science Foundation (IBN0212222 and IOB-0516789) to VFI and by grants from the Swedish Research Council (VR), the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) and the Swedish Foundation for International Co-operation in Research and Higher Education (STINT) to KG. JFS was the recipient of fellowships from the Brown and Hellmuth Hertz Foundations.

Supplementary Material Chromatin Immunoprecipitation (ChIP) Nuclear extracts were prepared as described by Ito et al., 1997 using MC, M1, M2 and M3 buffers, except that 0.3 g of floral inflorescence tissue was collected and powdered in MC buffer followed by direct fixation in M1 buffer supplemented with 1% formaldehyde. The immunoprecipitation of purified chromatin was performed using a ChIP assay kit (Upstate, Lake Pacid, NY, USA), according to the manufacturer’s instruction, and a commercially available monoclonal antibody against the HA tag (Santa Cruz Technology, CA, USA). Crude protein samples for quality control were separated in a NuPAGE TM 4–12% Bis-Tris gel (Invitrogen) and blotted on to a nitrocellulose membrane according to the manufacturer’s instructions. Protein filters were probed with the monoclonal HA antibody (see above) followed by enhanced chemiluminescent (ECL) detection (Amersham Biosciences, Amersham, UK). Fractions corresponding to gel, input, unbound and bound DNA samples were purified using the QIAquick PCR purification kit (QIAGEN GmbH, Hamburg, Germany). Gel samples were visualized on an ethidium bromide-stained 1% agarose (0.5 TBE) gel. Input and bound samples were analysed for the enrichment of specific DNA regions in the AP1 and AP3 promoter sequences using qRT-PCR. For reaction conditions and data treatment, see above. For primer sequences, see Table S4. The ratios of bound to input for each primer pair tested were normalized against the ratio of the transposon Mu-2, which serves as an internal control for non-specific binding of the HA antibody. Statistical analyses (Student’s t-test) were performed using http://graphpad.com/ quickcalcs/ttest1.cfm.

Chimeric shoot frequencies The presence of ap1-like flowers in primary inflorescences of PI–HA, PI–HA x cal-5, cal-5 and WT control plants was counted 10 days after bolting in two separate experiments. The cal-5 mutant was a gift from M. F. Yanofsky (University of California at San Diego, CA, USA). Genotyping was performed as described by Ferrandiz et al., 2000. Specimens examined by scanning electron microscopy (SEM) were mounted on stubs fresh, and examined at 20 kV in a Zeiss Supra 35 VP SEM (Carl Zeiss SMT, Oberkochen, Germany), with aperture 60 and working pressure 34 Pa. Brightness and contrast were adjusted using PHOTOSHOP (Adobe Inc., CA, USA). Statistical analyses (Student’s t-test) were performed using http://graph pad.com/quickcalcs/ttest1.cfm.

Acknowledgements We thank Drs Nicole Kubat and Toshiro Ito for assistance with the ChIP protocol and Stefan Gunnarsson and Gary Wife for help with

The following supplementary material is available for this article online: Appendix S1. Microarray data. Figure S1. Arabidopsis flowers expressing the 35S::PI-HA transgene produce sepals with petaloid margins. Table S1 Primers used for cloning of PISTILLATA cDNA Table S2 Primer sequences used in qRT-PCR experiments Table S3 Primers used to produce AP1 in situ probes Table S4 Primer sequences used in ChIP experiments This material is available as part of the online article from http:// www.blackwell-synergy.com

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