From the Institut de Biotechnologie des Plantes, CNRS UMR 8618, Université Paris-sud XI, 91405 Orsay, France
Received for publication, March 19, 2003 , and in revised form, May 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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A variety of HATs have been discovered (11). Many of them are related to yeast Gcn5, a member of two transcription regulatory 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. Moreover, 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.
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EXPERIMENTAL PROCEDURES |
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Genomic DNA and Total RNA Extraction, PCR, RT-PCR, and Northern BlotsArabidopsis leaves were used for genomic DNA extraction. PCR reactions were carried out by using the Promega TflI polymerase in the presence of 2% Me2SO. The primers used to check the T-DNA insertion in AtGCN5 were: 5'-CTGGTTGGACCCCAGATCAGTGGGGGC-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'-CTGCTTCGATTGACTCTCACTCTTCCC-3' (primer 1) and 5'-CGTGCATGTTGTTTCAAGTGGTTCATC-3' (primer 2); for the 3' fragment of AtGCN5, 5'-GCAATCACAGCAGATGAACAAG-3' (primer 3) and 5'-CCATCTTCTTCTATTGAGATTTAGCACCAG-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'-GCCGAGAGACAGCTTATTGC and 5'-AAGGATGCTGGATTTGATGC; for APETALA2 (AP2), 5'-AAGATATGCGGCTCAGGATG-3' and 5'-TCTGCAGCCAATTTTGATGA-3'; for APETALA3 (AP3), 5'-tgtttgggccactcaatatg and 5'-ggttctggtggaaacgaaga; for WUSCHEL (WUS), 5'-gtctgaactagctcttacgccggt-3' and 5'-tagccatgtctatggatctatgg-3'; for CLAVATA1 (CLV1), 5'-CGTGGAAGCTGCAAAGGCTTGTG and 5'-CCTGGGGTCAACAATCGCAACAAC; for SHOOT MERISTEMLESS1 (STM1), 5'-CTTTAAGCTCTCTATCCTCAGCTTG and 5'-GCCCATCATCACATCACATC; 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.
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Atgcn5 Cloning and ComplementationAtgcn5 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 0.5x 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 BlottingNuclear 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 StainingInflorescence were prefixed in 90% acetone at
room temperature for 20 min, rinsed in staining buffer without
5-bromo-4-chloro-3-indolyl--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 vacuum 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 AnalysesFor 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.
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RESULTS |
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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 1012 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, homeotic 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).
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AtGCN5 Is Required to Regulate the Floral Meristem Activity through the WUSCHEL/AGAMOUS PathwayCareful examination 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 1012 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).
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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).
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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 (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, suggesting 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).
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DISCUSSION |
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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). Gcn5 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 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.
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FOOTNOTES |
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To whom correspondence should be addressed. Fax: 33-1-69-15-34-25; E-mail:
zhou{at}ibp.u-psud.fr.
1 The abbreviations used are: HAT, histone acetyltransferase; RT, reverse
transcription.
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ACKNOWLEDGMENTS |
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REFERENCES |
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