# Arabidopsis Histone Acetyltransferase AtGCN5 Regulates the Floral Meristem Activity through the WUSCHEL/AGAMOUS Pathway

Claire Bertrand, Catherine Bergounioux, Séverine Domenichini, Marianne

Delarue, Dao-Xiu Zhou

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Claire Bertrand, Catherine Bergounioux, Séverine Domenichini, Marianne Delarue, Dao-Xiu Zhou. Arabidopsis Histone Acetyltransferase AtGCN5 Regulates the Floral Meristem Activity through the WUSCHEL/AGAMOUS Pathway. Journal of Biological Chemistry, 2003, 278 (30), pp.28246 - 28251. 10.1074/jbc.m302787200 . hal-05242274

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# Arabidopsis Histone Acetyltransferase AtGCN5 Regulates the Floral Meristem Activity through the WUSCHEL/AGAMOUS Pathway*

Received for publication, March 19, 2003, and in revised form, May 7, 2003
Published, JBC Papers in Press, May 9, 2003, DOI 10.1074/jbc.M302787200

Claire Bertrand, Catherine Bergounioux, Séverine Domenichini, Marianne Delarue, and Dao-Xiu Zhou‡

From the Institut de Biotechnologie des Plantes, CNRS UMR 8618, Université Paris-sud XI, 91405 Orsay, France

Histone acetyltransferases, which are able to acetylate histone and non-histone proteins, play important roles in gene regulation. Many histone acetyltransferases are related to yeast Gcn5, a component of two transcription regulatory complexes SAGA and ADA. In this work, by characterizing a mutation in the *Arabidopsis* GCN5 gene (*AtGCN5*) we studied the regulatory function of this gene in controlling floral meristem activity. We show that in addition to pleiotropic effects on plant development, this mutation also leads to the production of terminal flowers. The flowers show homeotic transformations of petals into stamens and sepals into filamentous structures and produce ectopic carpels. The phenotypes correlate to an expansion of the expression domains within floral meristems of the key regulatory genes *WUSCHEL* (*WUS*) and *AGAMOUS* (*AG*). These results suggest that *AtGCN5* is required to regulate the floral meristem activity through the *WUS/AG* pathway. This study brings new elements on the elucidation of specific developmental pathways regulated by *AtGCN5* and on the control mechanism of meristem regulatory gene expression.

The organization of chromatin structure provides a major barrier to gene transcription. In eukaryotic cells, there exist enzymatic activities to modify chromatin structure to control DNA accessibility. These activities include ATP-dependent chromatin remodeling complexes, represented by the Swi/Snf complexes (1), and the histone acetyltransferases (HAT)<fnref n="1" /> and histone deacetylases (2, 3). HATs and histone deacetylases can be targeted to promoters to activate or repress gene transcription; many regulated genes employ HATs to achieve transcriptional activation (4). Although an exact mechanism of how acetylation of histones contributes to transcriptional activation has not been known, it has been thought that acetylation of nucleosomal histones leads to relaxation of chromatin structure in such a way that transcription factors can gain access to chromosomal DNA (5). However, there are cases where increased histone acetylation correlates with repression (6, 7). In addition, acetylation of non-histone factors might have activating or repressing effects on transcription (8-10).

A variety of HATs have been discovered (11). Many of them are related to yeast Gcn5, a member of two transcription reg-

ulatory complexes, SAGA and ADA, within which Gcn5 serves as a nucleosomal HAT, capable of acetylating histone H3 in nucleosomes (12, 13). In yeast and animal cells, Gcn5 family members and CBP/p300 also acetylate non-histone proteins such as transcription factors (14, 9). Most HATs have a bromodomain (15), which is an acetyl-lysine binding motif found in a variety of chromatin-related proteins (16). Deletion mutations of GCN5 induce both up- and down-regulation of 4% of genes in yeast (17) and embryo growth defects in mice (18). The *Arabidopsis* GCN5 (AtGCN5) protein shows significant similarities to the HAT catalytic domains and bromodomains of previously described Gcn5 homologues (19). It has been recently reported that a T-DNA insertion in *AtGCN5* affects also about 5% of 8200 tested genes in vegetative tissues and produces pleiotropic effects on plant growth and development (20). This suggests that the function of AtGCN5 in gene regulation may be similar to that proposed for the Gcn5 family members and that plant Gcn5 proteins may be involved in the control of specific developmental pathways.

One of the plant characteristic developmental pathways is the floral organogenesis. Flowers contain four types of organs, sepals, petals, stamens, and carpels, that are arranged in four concentric whorls. The floral organ identity is specified combinatorially by three classes of homeotic genes, termed A, B, and C (21). Each class of the genes is active in two adjacent whorls: class A in whorls one and two, B in whorls two and three, and C in whorls three and four. One well studied example is the C function gene *AGAMOUS* (*AG*), which not only specifies stamen and carpel identity but also limits regeneration of floral meristem cells (21, 22). Proper temporal and spatial expression of *AG* is central to flower pattern formation. In wild-type plants, *AG* mRNA is expressed only in the central region of the floral meristem and in the inner two whorls of a developing flower (21, 22). This spatial and temporal expression pattern is regulated by a number of positive and negative regulators. The major repressor of *AG* in the first and second whorls is *APETALA2* (*AP2*). Loss of *AP2* function results in *AG* ectopic expression in the outer whorls and the corresponding homeotic transformations of these organs to carpels and stamens (23). *WUSCHEL* (*WUS*), a homeodomain protein that specifies stem cell identity and is expressed in a few cells in the center of shoot apical meristems (24), induces *AG* expression in the center of developing flowers (25, 26). In mature flowers, *AG* in turn represses *WUS* in mature flowers to terminate floral meristems (25, 26).

In this work we show a regulatory function of AtGCN5 in controlling floral meristem activity by characterizing a T-DNA insertion mutation of the gene. We show that in addition to pleiotropic effects on plant growth and development observed also in another mutation of the gene (20), this mutation induces the production of terminal flowers on the inflorescence. More-

<footnote>* This work was supported by Génoplante II Grants AF1999073 and AF2001019. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.</footnote>

<footnote>‡ To whom correspondence should be addressed. Fax: 33-1-69-15-34-25; E-mail:zhou@ibp.u-psud.fr.</footnote>

<footnote n="1">¹ The abbreviations used are: HAT, histone acetyltransferase; RT, reverse transcription.</footnote>
over, homeotic transformations of flower organs occur in the flowers. We show that the floral phenotypes correlate to alterations of the expression domains of WUS and AG within floral meristems. Our results suggest that *AtGCN5* is involved in the regulation of the spatial expression of key regulatory genes required for floral meristem function.

## EXPERIMENTAL PROCEDURES

*Plant Materials*—Arabidopsis thaliana plants were grown in a greenhouse under long day conditions (16 h of light) at 19.5 °C (day) and 17.5 °C (night). T-DNA insertion mutant plants (DLX8 from the Versailles collection) were in theWs background. The pAG-I:GUS and WUS:GUS transgenic lines were kindly provided respectively by L. Sieburth (27) and T. Laux (28) and were crossed withWs and DLX8 plants.

*Genomic DNA and Total RNA Extraction, PCR, RT-PCR, and Northern Blots*—Arabidopsis leaves were used for genomic DNA extraction. PCR reactions were carried out by using the Promega TfII polymerase in the presence of 2% Me₂SO. The primers used to check the T-DNA insertion in *AtGCN5* were: 5'-CTGGTTGGACCCAGATCAGTGGGGC-3', 5'-GGTATCGGGGAGTTGTAAGTTCTAC-3', which were specific for *AtGCN5*, and 5'-CTACAAATTGCCTTTTCTTATCGA-3', specific for the T-DNA sequence.

Total RNA was isolated from the leaves or flowers with the TRIzol reagents (Invitrogen). First strand cDNA was synthesized from 5 µg of total RNA using Superscript II reverse transcriptase (Invitrogen). For semi-quantitative PCR analysis, 2 µl of the reverse transcription were used per PCR in a final volume of 20 µl. The primers were as follows: for the 5' fragment of *AtGCN5*, 5'-CTGCTTCGATTGACTCTCACTCTTCC-3' (primer 1) and 5'-CGTGCATGTTGTTTCAAGTGGTTCATC-3' (primer 2); for the 3' fragment of *AtGCN5*, 5'-GCAATCACAGCAGATGAACAAG-3' (primer 3) and 5'-CCATCTTCTTCTATTGAGATTTAGCCAG-3' (primer 4), as indicated in Fig. 1A; for SUPERMAN, 5'-GAGCTTGCACATATGGAGAG-3' and 5'-CTGCCCTATATCTTGGTGAG-3'; for AGAMOUS, 5'-GTTGATTTGCATAACGATAACCAGA-3' and 5'-CACTGATACAACATTCATCGGAT-3'; for APETALA1 (AP1), 5'-GCCGAGAGACGCTTATTGC and 5'-AAGGATGCTGGATTTGATGC; for APETALA2 (AP2), 5'-AAGATATGCGGCTCAGGATG-3' and 5'-TCTGCAGCCAATTTTGATGA-3'; for APETALA3 (AP3), 5'-tgtttgggccactcaaatg and 5'-ggttctggtggaaacgaaga; for WUSCHEL (WUS), 5'-gtctgactagctcttacgccggt-3' and 5'-tagccatgtctatggatctgg-3'; for CLAVATA1 (CLV1), 5'-CGTGGAAGCTGCAAAGGCTTGTG and 5'-CCTGGGGTCAACAATCGCAACAAC; for SHOOT MERISTEMLESS1 (STM1), 5'-CTTTAAGCTCTCTATCCTCAGCTTG and 5'-GCCCATCATCACATCCATC; for actin 2, 5'-CTAAGCTCTCAAGATCAAAGGCTTA-3' and 5'-TTAACATTGCAAAGAGTTTCAAGGT-3'. The PCR products were electrophoresed, blotted onto a Hybond N+ membrane (Amersham Biosciences), and hybridized with ³²P-labeled probes made by using the Random Primer kit (Appligene). For Northern blots, 10 µg of total RNA were separated by electrophoresis in a denaturing agarose gel, blotted, and hybridized with ³²P-labeled PCR products.

*Atgcn5 Cloning and Complementation*—Atgcn5 cDNA was amplified as two fragments by RT-PCR from Ws leaf mRNA by using the primer sets as described above. The PCR fragments were cloned into pGEM-T (Promega) and sequenced. The fragments were assembled by using a unique KpnI site present in the overlapping region of the fragments. The full-length cDNA was further cloned under the control of the cauliflower mosaic virus 35 S promoter (35S:*AtGCN5*) and introduced into Agrobacterium tumefaciens (HBA10S). DLX8 plants were transformed using the floral dip method (29). Seeds from the T1 plants were selected on 0.5× Murashige and Skoog medium containing 50 mg/liter gentamycin. Gentamycin-resistant plantlets were transferred to soil and grown in the greenhouse under long day conditions. The presence of the 35S:*AtGCN5* insertion was checked by PCR.

*Western Blotting*—Nuclear proteins were isolated from rosette leaves, resolved by SDS-PAGE, transferred to nitrocellulose membrane, and incubated first with a polyclonal rabbit anti-acetylated histone H3 antibody (Chemicon International) and then with a goat anti-rabbit IgG peroxidase-conjugated antibody (Bio-Rad). Detection of the target proteins was performed by chemiluminescence using ECL⁺ System (Amersham Biosciences). The bands were scanned, and their densities were quantified at >50 locations.

*GUS Staining*—Inflorescence were prefixed in 90% acetone at room temperature for 20 min, rinsed in staining buffer without 5-bromo-4-chloro-3-indolyl-β-D-glucoronic acid (X-Gluc), infiltrated with staining solution (100 mM sodium phosphate buffer, pH 7, 5 mM potassium ferrocyanide, 5 mM potassium-ferricyanide, 1 mM X-Gluc) under a vac-

<figure><img src="image_3.png" /></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>**FIG. 1. Characterization of a T-DNA insertion mutation within AtGCN5.** A, schematic representation of the T-DNA insertion in the AtGCN5 gene. Exons (open boxes) encoding the HAT domain (dashed underline) and the bromodomain (solid underline) are indicated. The arrow indicates the 5' end of the gene. The filled triangles represent the three PCR primers used to confirm the insertion (see "Experimental Procedures"). The location of four primers used in RT-PCR are indicated by small arrows. B, RT-PCR analysis of AtGCN5 expression in homozygous insertion mutant (DLX8) and wild-type (WT) inflorescence with primer pairs 1, 2 and 3, 4 as indicated in A. C, Western blot analysis of 10 (1), 20 (2), and 5 µg (3) of nuclear proteins extracted from DLX8 or wild-type rosette leaves with anti histone H3. The arrow and the asterisk indicate the acetylated H3 and a cross-reacted band, respectively. The bands were scanned and quantified from more than 50 locations within each band. The bars represent relative average values with the strongest band at 100. The S.D. are indicated.</figcaption></figure>

uum for 15 min, and incubated at 37 °C for 14 h. After a progressive dehydration in a series ethanol concentrations up to 70%, samples were fixed and embedded in paraplast plus (Sherwood Medical Corp., St. Louis, MO). Eight-µm sections were performed and observed under a Zeiss light microscope.

*Scanning Electron Microscope Analyses*—For scanning electron microscope (Hitachi S-3000) analysis, samples were slowly frozen at -18 °C under a partial vacuum on the Peltier stage before observation under the environmental secondary electron detector mode.

## RESULTS

*Inactivation of AtGCN5 Results in Pleiotropic Effects on Plant Development*—Searching for insertion mutations within *Arabidopsis* HAT genes led to the identification of a T-DNA insertion line, DLX8, from the Versailles collection. The insertion site was located in the 10th intron of *AtGCN5*, disrupting the bromodomain-coding region (Fig. 1A). This mutant line seemed to contain a single insertion due to offspring segregating three to one for kanamycin resistance. A third of kanamycin-resistant plants showed serious growth defects with a
<figure><img src="image_6.png" /><figcaption>FIG. 2. Pleiotropic phenotypes induced by the T-DNA insertion within AtGCN5. A, comparison between a wild-type plant (left) and a homozygous DLX8 plant (middle) at the age of 5 weeks. A mature DLX8 plant is on the right. B, comparison between wild-type (a–d) and DLX8 (e–h) rosette leaves (a and e, bars, 0.5 cm), early produced flowers (b and f, bars, 1 mm), siliques (c and g, bars, 1 cm), and seeds (d and h, bars, 1 mm).</figcaption></figure>

smaller plant size and loss of apical dominance during later
growth stages (Fig. 2A). PCR results confirmed that plants
showing this phenotype corresponded to homozygous T-DNA
insertion mutants *AtGCN5* (not shown). The wild-type pheno-
type was restored when the mutant plants were complemented
by transformation with a 35S::*GCN5* construct (not shown).
High levels of *AtGCN5* mRNA were detected in different organs
of the wild-type plants (Ref. 19 and data not shown). RT-PCR
experiments with a primer set corresponding to the bromodo-
main region of the gene showed no *AtGCN5* expression in the
homozygous mutant plants. A weaker band was amplified from
the same RNA samples when a primer pair corresponding to
sequences before the T-DNA insertion was used (Fig. 1B). This
suggests that a hybrid RNA with T-DNA sequences was pro-
duced but stably accumulated. The acetylation level of histone
H3 was compared between the wild-type and the mutant plants

by Western blots using antibodies against acetylated histone
H3. The results showed a reduction of histone H3 acetylation in
the mutant plants (Fig. 1C), indicating that *AtGCN5* is in-
volved *in vivo* in histone H3 acetylation and that other acetyl-
transferases are also implicated in the acetylation.

The mutation strongly affected the leaf morphology. Rosette
leaf expansion was reduced, leading to smaller and upward-
curled rosette leaves with slight serrations of leaf margins (Fig.
2B). The first 10–12 flowers produced from an inflorescence
had a disorganized structure, although the overall size of those
flowers was not affected (Fig. 2B). In late-arising flowers, ho-
meotic transformation of flower organs was observed (see Fig.
5 and next paragraph). The DLX8 plants were partially fertile,
producing few and deformed siliques and seeds (Fig. 2B).

*AtGCN5 Is Required to Regulate the Floral Meristem Activity*
*through the WUSCHEL/AGAMOUS Pathway*—Careful exam-
<caption>TABLE I
Average stamen numbers per flower</caption>

Average stamen numbers were calculated from 30 early arising DLX8 or wild-type flowers. Values are ± S.D.

<table><thead><tr><th></th><th>DLX8</th><th>Wild type</th></tr></thead><tbody><tr><td>Stamens/flower</td><td>6.9 ± 0.9</td><td>5.7 ± 0.5</td></tr></tbody></table>

<figure><img src="image_7.png" /><figcaption>FIG. 3. Expression of developmental regulatory genes in DLX8 or wild-type (WT) plants. A, Northern blot analysis of total RNA extracted from rosette leaves or early produced flowers with probes as indicated. B, RT-PCR analysis of flower RNA with primer sets specific to the genes indicated on the left.</figcaption></figure>

ination of early produced mutant flowers found that the identity of sepals in whorl one, petals in whorl two, stamens in whorl three, and carpels in whorl four was not altered (Fig. 2B) except that an increase in stamen number per flower was observed (Table I). After the production of about 10–12 flowers, the inflorescence gave rise to flowers in which the sepals of whorl one were not present. The organs produced in this whorl either had a filamentous structure or aborted during growth (see Fig. 4A). Organs in whorl two were transformed into stamens, consequently producing supernumerary stamens (see Fig. 4A). Extra carpel-like organs were observed in some flowers (see Fig. 4, A and B). The aberrant terminal flowers appeared at apices of inflorescence branches, leading to termination of the inflorescence meristem (see Fig. 4, C and D). In addition, the growth of other flowers previously produced within the same inflorescence was inhibited or aborted (see Fig. 4A).

The terminal flower phenotypes were reminiscent of plants overexpressing *AG* under the cauliflower mosaic virus 35 S promoter (30). We therefore examined the expression levels of *AG* in DLX8 flowers by RT-PCR and RNA blots and found that the expression levels of *AG* were increased in flowers of the mutant plants (Fig. 3).

To examine whether the *AG* expression domain was altered or not, we crossed a *pAG-I::GUS* transgenic line (27) with DLX8 and wild-type *Ws* ecotype plants. F2 inflorescence from the crosses were stained to detect GUS expression. The mutant flowers were more darkly stained than the wild-type transgenic flowers (see Fig. 5, A and B). GUS expression could be seen throughout the terminal flower in DLX8 (see Fig. 5C). In fact, the *AG* promoter activity was detectable in the primordia of the outer whorls of the terminal flower (see Fig. 5D), suggesting that *AG* was activated early during floral organogenesis in the mutant inflorescence. In the non-terminal flowers of the mutant, the *AG* promoter activity in the mutant was detected not only in whorls three and four as observed in the wild-type flowers (see Fig. 5E) but also in the base of sepals and petals

<figure><img src="image_8.png" /><figcaption>FIG. 4. Phenotypes of DLX8 flowers. A, terminal flowers in inflorescence branches. Filamentous organs in the first whorl (arrowheads), ectopic carpel-like organs (asterisks), and suppressed flower buds (arrows) are indicated. *Bar*, 1 mm. *B*, enlarged view of ectopic carpel-like organs. *Bar*, 0.2 mm. *C*, comparison of wild-type (*left*) and DLX8 (*right*) inflorescence. *Bar*, 1 mm. *D*, scanning electron microscopy of wild-type (*left*) and DLX8 (*right*) inflorescence. *Bar*, 100 µm.</figcaption></figure>

(whorls one and two) (see Fig. 5F). The GUS expression domain was extended to the pedicel of the flowers (see Fig. 5F). These observations indicate that the expression domain of *AG* was enlarged in DLX8 plants.

Because in wild-type plants *WUS* activates *AG* expression in the center of developing flowers and misexpression of *WUS* in developing flowers under the control of floral-stage-specific promoters induced similar but more severe phenotypes (25, 26), we therefore examined the expression of *WUS* in DLX8. Using RT-PCR, we detected a clear increase of *WUS* expression in the mutant flowers (Fig. 3). In wild-type plants, *WUS* is expressed in a few cells in the inner region of the shoot meristem. To examine the expression domain of *WUS* in the DLX8 plants, we crossed a *WUS::GUS* transgenic line (28) with DLX8 and wild-type *Ws* ecotype plants. F2 young inflorescence from the crosses were stained to detect GUS activity. In the wild-type background, the GUS activity was relatively weak and detected in a few cells within the floral meristems (see Fig. 5, G and I). In the DLX8 background, the GUS activity was higher than the wild type (compare Fig. 5, H and G) and was detected throughout the apical meristem from the primary inflorescence, even in the primordia of the first whorls of developing flowers (Fig. 5J). However, no GUS activity was detected in the mature flowers (not shown). This enlargement of the expression domain of the *WUS* promoter correlates to that of *AG* in DLX8 flowers, sug-
gesting that the induction of *AG* expression in the floral outer whorls may be due to the expansion of *WUS* expression in the mutant.

Because the major repressor of *AG* in the first and second whorls is *AP2* that is expressed throughout the floral meristem (23), we therefore examined the expression level of *AP2* in DLX8. We found that the mRNA level of *AP2* as well as another A function gene *APETALA* 1 (*AP1*) (31) in DLX8 flowers was slightly reduced when compared with that in the wild-type plants (Fig. 3). The expression of a B-function gene *APETALA3* (*AP3*) was not significantly changed (not shown). Inhibition of histone deacetylation by antisense expression of AtHD1 induces ectopic expression of *SUPERMAN* (*SUP*) (32), which controls floral whorl boundaries (33). However, the mRNA level of *SUP*, was not affected in DLX8 (Fig. 3). To know whether *AtGNC5* affects the expression of shoot apical meristem regulatory genes, we checked the mRNA levels of *SHOOT MERISTEMLESS* (*STM*) (34) and *CLAVATA 1* (*CLV1*) (35) in DLX8 plants. No significant change was detected when compared with wild-type plants (Fig. 3). This is consistent with the normal appearance of shoot apical meristems observed in DLX8 (not shown).

DISCUSSION

The recessive nature of the DLX8 mutation and the complementation experiments indicate that this is a loss of function mutation. DLX8 plants may still produce a low level of a truncated AtGCN5 protein. However, without the bromodomain, this protein would be unable to bind to promoter nucleosomes. This is consistent with the lower levels of H3 acetylation in the mutant. In addition, a mutant allele of *AtGCN5* showing pleiotropic phenotypes has been recently published (20). It is, therefore, unlikely that the phenotype we observed is due to any dominant effect of the truncated protein, although it is not excluded that the truncated protein retains some function.

The ubiquitous expression pattern of *AtGCN5* and the pleiotropic effects of its mutation suggest that it functions in the regulation of many genes required for different biological processes. The mutation effects might be induced as a consequence of the reduction of histone H3 acetylation. Whether more dramatic reduction of histone acetylation levels induces severer phenotypes or affects more genes awaits the analysis of an allelic series of *AtGCN5* mutants and a multiple knockout of several HAT genes. Histone acetylation/deacetylation appears to be a general regulatory mechanism responsible for controlling plant development. Blocking histone deacetylation by antisense expression of *AtHD1* also induces pleiotropic effects in transgenic plants (32). Gen5 homologues in yeast and animal cells are usually recruited to promoters via protein interactions with promoter-specific transcription factors. In yeast this acetyltransferase is required for the expression of a subset (4%) of genes (17). Recent analysis by DNA microarray shows that the mutation of *AtGCN5* induces expression changes of about 5% of the tested 8200 genes in *Arabidopsis* aerial vegetative tissues, with 75% of the genes up-regulated and 25% down-regulated (20). Changes in gene expression induced by the mutation of *AtGNC5* in other stages of development or organs such as the flower is not known. Similar approaches would be helpful to detect changes of gene expression in the floral meristem induced by the mutation of *AtGCN5*.

We have shown that the DLX8 mutation induces the production of terminal flowers and floral organ homeotic transformations (Fig. 4). However, in the work reported by Vlachonasios et al. (20), there is no description of any terminal flower or organ transformation in their mutant allele, although aberrant early formed flowers have been shown. One explanation might be that their T-DNA insertion is in the last exon of the gene, which

<figure><img src="image_9.png" /><figcaption>FIG. 5. **AG and WUS expression profiles in DLX8 flowers and inflorescence.** A–F, *pAG-I*:GUS expression profiles. A, a wild-type inflorescence; *B*, a determinate DLX8 inflorescence; the terminal flower is indicated (arrow). *C*, a DLX8 terminal flower with blue staining of all organs. *D*, a section of a DLX8 determinate inflorescence meristem. Staining of the primordia from the outer whorls is indicated (chevrons). *E*, wild-type flower buds with blue staining of the developing organs (stamens and carpels) from the inner whorls. *F*, DLX8 non-terminal flower buds with staining on the base of petals and sepals (indicated by arrowheads). *G–J*, WUS::GUS expression profiles. *G*, GUS staining of the wild-type primary inflorescence. *H*, GUS staining of the DLX8 primary inflorescence. *I*, a section of the wild-type inflorescence with blue staining of inner cells of the apical meristems. *J*, a section of the DLX8 primary inflorescence with blue staining throughout the meristems and flower buds. *IM*, inflorescence meristem. *F*, flower bud. *Bars*: A–C, *G*, and *H*, 0.2 mm; *D–F*, 100 µm; *I* and *J*, 50 µm.</figcaption></figure>

may be less severe than the DLX8 insertion that disrupts the coding region of the bromodomain. Consistent with the floral phenotypes that we have observed in DLX8 plants, the expression domain of *AG* is expanded to the outer whorls of the floral organs. We have found that among a few known regulators of *AG*, *WUS* expression domain that is restricted normally to a few cells in the apical meristems is largely expanded in floral and inflorescence meristems. Therefore, it is likely that the induction of *AG* expression is a consequence of the expansion of the *WUS* expression domain in DLX8. Higher levels of *AG* in turn repress *WUS* to terminate the inflorescence meristems in
DLX8. A slight reduction of A function gene (AP1 and AP2) expression may be a consequence of the increased expression of AG that is antagonistic to A function genes.

This indicates that *AtGCN5* is required to control floral meristem regulatory gene expression, which possibly restricts the *WUS* expression domain within the floral meristem. We noted that the misexpression of *WUS* and *AG* was detected not only in the terminal flowers but also in more precociously produced flowers (Fig. 5). It is not known why these flowers do not show homeotic transformation but show supernumerary stamens (Table I). One possibility may be that during the growth misexpressed *WUS* or *AG* protein accumulates to a threshold level, leading to homeotic phenotypes.

Histone acetyltransferases are usually required for gene activation. However, direct repression of gene expression by Gcn5 family members has been documented (10). The observation that the two floral meristem genes show increased expression in the mutants indicates that AtGCN5 plays a negative role on the expression of the genes. Our results can not distinguish between the direct and indirect effect of AtGNC5 on the expression of the genes. One possibility is that the mutation of *AtGCN5* reduced the expression of a repressor of *WUS*. Alternatively, AtGCN5 might act directly on the promoter of *WUS*, recruiting a repressor of *WUS* transcription. In summary, our results indicate that AtGCN5 is involved in the control of a specific plant development pathway by regulating the expression domains of *WUS* and *AG* in the floral meristem.

*Acknowledgments*—We thank Dr. T. Laux for providing the *WUS::GUS* transgenic line, Dr. L. Sieburth for the *pAG-I::GUS* line, Roland Boyer for the photographs, and Dr. R. Stevens for reading the manuscript.

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