Genes and functions controlled by floral organ identity genes

Seminars in Cell & Developmental Biology 21 (2010) 94–99

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Review

Genes and functions controlled by floral organ identity genes Robert Sablowski ∗ Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, United Kingdom

a r t i c l e

i n f o

Article history: Available online 4 September 2009 Keywords: Floral homeotic genes Target genes Flower development

a b s t r a c t Floral organ identity genes specify the identity of floral organs in a manner analogous to the specification of body segments by Hox genes in animals. Different combinations of organ identity genes co-ordinate the expression of genes required for the development of each type of floral organ, from organ initiation until final differentiation. Here, I review what is known about the genes and functions subordinate to the organ identity genes. The sets of target genes change as organ development progresses and ultimately organ identity genes modify the expression of thousands of genes with a multitude of predicted functions, particularly in reproductive organs. However, genes involved in transcriptional control and hormone functions feature prominently among the early and direct targets. Functional analysis showed that control of organ-specific tissues and structures can be delegated to specialised intermediate regulators, but organ identity genes also fine-tune genes with general roles in shoot organ development, consistent with the notion that organ identity genes modify a core leaf-like developmental program. Future challenges include obtaining data with cellular resolution, predictive modelling of the regulatory network, and quantitative analysis of how organ identity genes and their targets control cell behaviour and ultimately organ shape. © 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Identifying the targets of floral organ identity genes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Functional analysis of individual targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Combinatorial action and the cis-regulatory code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and future challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Note added in proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

Abbreviations: AG, AGAMOUS; AGL5, AGAMOUS-like 5; AP1, APETALA1; AP3, APETALA3; ARF, auxin response factor; ATH1, ARABIDOPSIS THALIANA HOMEOBOX GENE1; ATX1-2, ARABIDOPSIS TRITHORAX1-2; bHLH, basic helix-loop-helix; bZIP, basic leucine zipper; ChIP, chromatin immunoprecipitation; ChIP-chip, ChIP followed by oligonucleotide chip analysis; ChIP-seq, ChIP followed by deep sequencing of immunoprecipitated DNA; DAD1, DEFECTIVE IN ANTHER DEHISCENCE1; DEF, DEFICIENS; GA4, GA-REQUIRING 4; GFP, green fluorescent protein; GNC, GATA, NITRATE INDUCIBLE, CARBON METABOLISM-INVOLVED; GNL, GNC-LIKE; GUS, ␤-glucuronidase; Hox, homeobox; JA, jasmonic acid; JAG, JAGGED; MADS, MCM1, AG, DEF, SRF; MPSS, massively parallel sequence signature; NAC, NAM, ATAF1-2, CUC1-2; NAP, NAM-LIKE, ACTIVATED BY AP3/PI; NUB, NUBBIN; PHA-4, pharynx-4; PI, PISTILLATA; SEP1/2/3/4, SEPALLATA 1/2/3/4; SHP1/2, SHATTERPROOF1/2; SPL, SPOROCYTELESS; STY1/2, STYLISH1/2; TCP, TB1, CYC, PCF1-2. ∗ Tel.: +44 1603 450530; fax: +44 1603 450045. E-mail address: [email protected]. 1084-9521/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2009.08.008

The control of floral organ identity is one of the most striking examples of how regulatory genes determine plant structure (reviewed by [1,2]). Each of the four types of floral organs (sepals, petals, stamens and carpels) is specified by a unique combination of regulatory genes. In Arabidopsis, sepal development is guided by APETALA1 (AP1) combined with any of four SEPALLATA (SEP1–SEP4) genes; petals are specified by AP1, SEP1–3, APETALA3 (AP3) and PISTILLATA (PI); stamens develop under the control of AP3, PI, SEP1–3 and AGAMOUS (AG), while the combination of only AG and SEP1–3 directs carpel formation. These gene combinations are not only necessary, but sufficient for the development of floral organs: if the required set is artificially expressed outside flowers, leaves are transformed into the corresponding floral organs [3,4]. Conversely, in mutants that are unable to specify any type of floral organ, flowers are made of leaf-like organs [5,6]. These results are consistent

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with the idea proposed by Goethe more than 200 years ago that floral organs and leaves are variations of the same basic type of organ [2]. The ability of floral organ identity genes to replace one organ type with another and their combinatorial action mirror the function of Hox genes, which specify the identity of body segments in animals [7,8]. Another similarity with Hox genes is that the floral organ identity genes control all stages of development of the body parts they specify, from initiation, through morphogenesis to cell differentiation [9,10]. In both cases, expression patterns are maintained throughout development by auto-regulation and by chromatin modification. Both Hox and floral organ identity genes encode transcription factors, but belong to unrelated families (homeodomain and MADS families, respectively). Thus the parallels between organ identity and Hox genes are a clear example of convergent evolution, with similar developmental strategies executed by unrelated sets of genes [11]. The combinatorial action of floral organ identity genes is reflected by interactions between the encoded proteins [12,13]. Therefore it is believed that different complexes of MADS proteins are able to activate or repress the sets of genes required for the development of each type of floral organ. However, as in the case of Hox genes and in fact throughout developmental biology, a large unexplained gap remains between the molecular function of these transcriptional regulators and their striking phenotypic effects. To begin to understand how the activity of floral organ identity genes is translated into the cellular activities that actually build floral organs, we need to reveal the gene expression programme that is co-ordinated by these genes. Specific questions include: - What kinds of genes and functions are controlled? Do the organ identity genes directly control genes involved in basic cellular functions, such as division, expansion and metabolism, or are these genes controlled indirectly through networks of regulatory genes and signalling molecules? - How do organ identity genes modify the underlying leaf-like gene expression program? Do they activate sets of organ-specific of genes or do they modify the activity of genes with roles in multiple organs (for example, genes required for the differentiation of cell types that are common between organs)? - How are different sets of target genes selected in different places and times? Much of the answer to this depends on understanding how organ identity genes function in combination with each other, with other transcription factors and with target promoters. Here, I review our progress in identifying the targets of floral organ identity genes and how this has contributed to answering the questions above. 2. Identifying the targets of floral organ identity genes. In the early days, candidate target genes were revealed by expression patterns or by low-throughput differential expression screens. The first example was AGL5 (AGAMOUS-like 5, subsequently re-named SHATTERPROOF2, SHP2), which was identified as a target of AG because it was expressed specifically in carpels and not expressed in the ag mutant; furthermore, AG bound in vitro to the SHP2 promoter and ectopic AG activated a SHP2:GUS reporter gene [14]. The first evidence of direct regulation in vivo came from a screen for changes in the floral mRNA population after posttranslational activation of AP3, with indirect effects blocked by cycloheximide [15]. In this screen, NAP (NAM-related, activated by AP3/PI), which encodes a member of the NAC family of transcription factors, was identified as an immediate target of AP3 and PI during petal and stamen development.

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The subsequent sequencing of the Arabidopsis genome and development of expression arrays allowed analysis of gene expression at a much larger scale. The most straightforward approach was to compare gene expression in different floral organ identity mutants. Zik and Irish used cDNA arrays covering about a fourth of all Arabidopsis genes to identify a set of genes downstream of AP3/PI, which was enriched for genes involved in stress responses and cell wall metabolism [16]. Wellmer et al. [17] used an array of floral cDNAs and a genome-wide oligonucleotide array to compare a wider range of mutants with organ identity changes. Their experiments revealed a small number of transcripts enriched in sepals (13) or petals (18), but a much larger set of genes expressed specifically in carpels (206) or stamens (1162), many of which are related to gametophyte development [18]. Genes involved in general cellular maintenance (DNA recombination, protein synthesis, protein folding) were under-represented, while functional classes such as embryonic development and cell wall modification were overrepresented [17]. Massively parallel sequence signatures (MPSS) were also used to compare the transcriptomes of mutant flowers with wild-type flowers and vegetative tissues [19]. The stamenenriched set identified by MPSS showed good agreement with the array experiments, but the overlap for other organ types was small; these discrepancies may result from the different criteria used to define organ-enrichment (mutants compared, baseline expression, statistical analysis). However, two common themes emerged from all experiments comparing gene expression in different organ types. First, the organ identity genes directly or indirectly influence a wide array of developmental and cellular processes. Second, the reproductive organs clearly have more specilaised developmental programs than perianth organs. The experiments described above were concerned only with spatial differences in gene expression and corresponded mostly to late stages of organ development. Other papers were concerned with temporal changes in the transcriptome. Bey et al. [20] analysed gene expression during the final stages of sepal and petal development in Antirrhinum and used a temperature-sensitive allele of DEFICIENS (DEF, the snapdragon orthologue of AP3) to detect genes that responded rapidly after DEF was activated. They noted that 60% of differentially expressed genes were stage-specific and that at late stages of petal development DEF appears to mostly regulate genes involved in metabolism and cell differentiation. In contrast, a disproportionate number of genes preferentially activated during early bud development in Arabidopsis and rice encode transcription factors [21–24]. Genes involved in the synthesis and response to hormones (gibberellin, auxin) were also over-represented in the transcriptome of early buds [21–23]. The overall conclusions of these time course experiments were that the gene expression programme under the floral organ identity genes changes over time and that early stages include a large proportion of regulatory genes (Fig. 1). The larger number of genes with metabolic and transport functions expressed at later stages of development could reflect a change in the types of functions controlled by organ identity genes, or it could reflect the accumulation of indirect effects on gene expression. To distinguish between these possibilities, it was necessary to identify the direct targets of organ identity genes. Although rapid response to a regulatory gene (as in the temperature shift experiments described above for DEF) is suggestive, proof of direct interaction requires chromatin immunoprecipitation (ChIP). Gomez-Mena et al. [21] used ChIP to confirm that the AG protein binds directly to some of its early target genes. This included AG itself, AP3 and SEP3, showing that an auto-regulatory loop maintains expression of the organ identity proteins that are predicted to function as a multiprotein complex. More recently, a number of important insights came from using ChIP-chip and ChIP-seq to obtain a global view of the direct targets of SEP3 in the wild type and in the ag mutant [25]. SEP3 bound in vivo to a large number of

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Fig. 1. Frequency of molecular and biological functions attributed to genes expressed at early and late stages of reproductive organ development, compared with the genomewide frequencies. Early stamens/carpels refers to the combined set of 149 genes activated by AG at 1, 3 and 7 days after organ initiation according to Gomez-Mena et al. [21]; late stamens/carpels correspond to the combined sets of 1422 genes preferentially expressed in stamens or carpels according to Wellmer and co-workers [18]. The pie charts show the frequency of terms found within the gene annotations for each set, belonging to the gene ontology (GO) categories shown on the left. GO analysis was performed through the TAIR web page (http://arabidopsis.org/tools/bulk/go/index.jsp), except that some molecular function categories were combined (“transcription factor activity”, “nucleic acid binding”, “DNA or RNA binding” all combined as “transcription factor activity, DNA or RNA binding”; “transferase activity”, “hydrolase activity” and “other enzyme activity” all combined as “hydrolase activity, transferase activity, other enzyme activity”).

sites (3457 loci, or 13% of Arabidopsis genes), including many of the previously identified targets of organ identity genes (e.g. SEP3, AG, AP3, SHP1, SHP2 and NAP). Binding to SEP3 was usually within a few hundred bp upstream or downstream of coding sequences, 72% of which were differentially expressed during flower development or in floral homeotic mutants, suggesting that the majority of SEP3 targets were functionally relevant. There was a large overlap between sites bound in the wt and in the ag mutant, suggesting that many SEP3 targets are shared between perianth and reproductive organs, although the regulatory outcome (activation or repression) might vary in different organs. Finally, gene ontology analysis revealed that the direct targets of SEP3 had a functional bias similar to that found in the transcriptome of early buds, with enrichment for genes encoding transcription factors, genes involved in the synthesis and response to hormones (particularly auxin) and in lipid metabolism. In summary, early and direct targets of organ identity targets are enriched for genes encoding transcription factors and include the organ identity genes themselves, whose expression is maintained by auto-regulatory loops. Genes involved in hormone synthesis and responses also feature prominently among the target genes. The sets of targets change as development progresses and ultimately organ identity genes modify the expression of thousands of genes, particularly in reproductive organs. 3. Functional analysis of individual targets From the multitude of target genes identified, a small subset has been selected for in-depth functional analysis. An early example was the AG target gene SHP2, which functions redundantly with its close homolog SHP1 in regulating the development of specific tissues of the carpel that are subsequently required for releasing seeds from the fruits [26]. This began to reveal how specific aspects of organ development are delegated from the organ identity genes to intermediate regulators. Another example is SPOROCYTELESS (SPL), which encodes a putative transcription factor required for both male and female microsporogenesis [27] and was identified as a direct target of AG and SEP3 [25,28]. When activation by AG was

bypassed using a ubiquitously inducible version of SPL, sporogenesis was seen in petals, showing that SPL expression was sufficient to transfer a subset of the developmental program of reproductive organs to perianth organs. STYLISH1 (STY1) is a direct target of SEP3 and AG [25] and encodes a RING finger protein that together with the close homologue STY2 is required for development of the apical tissues of the carpel (style and stigma) [29]. Artificial expression of STY1/2 throughout the carpel caused ectopic development of style cells, showing that STY1/2 are master regulators of a specific subset of carpel tissues. These examples showed that the developmental program downstream of organ identity genes is at least in part modular, with the development of discrete, organ-specific structures under the control of specialised, intermediate regulators. However, organ identity genes also modulate the expression of genes with more general roles in the development of shoot organs. NUBBIN (NUB) and JAGGED (JAG) encode closely related zinc finger proteins that have been implicated in the regional control of cell division and organ growth [30–32]. NUB has been identified as a direct target of AG, while JAG is bound in vivo by SEP3 [21,25]. jag mutant flowers show truncated growth of sepals and petals, but only mild defects in reproductive organs, where JAG is also expressed. This is because in stamens and carpels JAG functions redundantly with NUB: the jag nub double mutant has severe defects in the growth of distal regions of stamens and carpels. Thus SEP3 and AG directly control genes with redundant roles in organ growth: JAG, which functions in all shoot organs, and NUB, which within the flower has become more specialised for reproductive organs. ATH1 is another gene with a widespread role in organ development that is directly targeted by AG and SEP3 [21,25]. ATH1 encodes a BELL-type homeodomain protein that controls the development of the basal region of shoot organs, particularly at the boundary between organs and the stem (or the receptacle in the flower). The most obvious phenotype of the ath1 mutant, however, is that the stamens fails to abscise—so ATH1 function has become particularly limiting at the base of one the organs specified by AG [33]. A third case of target genes with general functions in shoot organs is the regulation by AP3/PI of GATA, NITRATE INDUCIBLE,

R. Sablowski / Seminars in Cell & Developmental Biology 21 (2010) 94–99

Fig. 2. Floral organ identity regulators modify the basic organ development programme and activate targets that control the development of specialised tissues and structures of floral organs. The figure uses as an example the combination of organ identity proteins that promote stamen development (AP3, PI, AG and SEP3). These are shown as a multiprotein complex that regulates genes with general roles in shoot organ development (ATH1, JAG/NUB) and genes that control specialised features of stamen development (SPL, DAD1). Arrows indicate activation, blunted line represents repression. X, Y, W, Z represent hypothetical uncharacterised targets.

CARBON METABOLISM-INVOLVED (GNC) and GNC-LIKE (GNL), which encode GATA transcription factors that regulate carbon and nitrogen metabolism in leaves [34,35]. Phytohormones are another type of molecule with versatile roles in development whose function is controlled locally by organ identity genes. For example, jasmonic acid (JA) is required for different aspects of root, flower and fruit development, in the latter including pollen maturation, anther dehiscence and filament elongation. The key role of JA in stamens is also indicated by the substantial number of JA-regulated genes expressed during stamen development [18,36]. One of the roles of AG in stamens is to activate JA production: DEFECTIVE IN ANTHER DEHISCENCE1 (DAD1), which encodes the first enzyme in JA biosynthesis, is a direct target of AG during late stages of stamen development [37]. During early flower development, AG also directly activates GA-REQUIRING4 (GA4), which encodes a key gibberellin biosynthetic enzyme [21]. The functional significance of this remains unknown, although one possibility is that gibberellin could function in a positive feedback loop to reinforce expression of organ identity genes [38]. A further link between organ identity genes and hormone function is indicated by the large number of SEP3 targets involved in signalling and homeostasis of auxin, gibberellin and brassinosteroids [25]. The same authors showed that binding sequences for the auxin response factors (ARFs) are enriched in the vicinity of SEP3 binding sites and that a dominant repressive version of SEP3 causes floral defects similar to those of auxin signalling mutants, suggesting that SEP3 and ARFs could co-operate in the regulation of auxin-responsive genes [25]. In conclusion, organ identity genes activate intermediate regulators, which direct the development of specialised tissues and structures of floral organs. Additionally, the organ identity proteins fine-tune the activity of genes with general roles in organ development (Fig. 2). The latter is consistent with the idea mentioned above, that floral organs and leaves are variations of the same ancestral type of organ, and therefore must share a core developmental program.

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torial action of organ identity proteins is the quartet model [13], according to which the MADS proteins function in multiprotein complexes, each containing the set of organ identity proteins that specify a particular organ type. It is puzzling, however, that in spite of their very distinct functions in vivo, the MADS proteins bind in vitro to very similar DNA sequences, collectively called CArG boxes and typically matching the consensus CC(AT)6 GG. The ability of each complex to select specific target genes could arise from differences in affinity for specific CArG box sequences, from co-operation with additional, spatially restricted transcription factors, or from a combination of both. Biases in CArG boxes or the presence of binding sites for co-factors might be detected by comparing large sets of promoters bound by each type of MADS complex. When searching the sequences targeted in vivo by organ identity proteins, the usual starting point to look for CArG boxes. The CArG consensus sequence is expected to occur frequently by chance, so finding it in a given promoter is hardly evidence that it is targeted by MADS proteins [39]. Enrichment for CArG boxes within a set of genes, however, could be evidence of regulation by MADS proteins. Sets of genes expressed at relatively late stages in different organ types were not enriched for CArG boxes [17,19], possibly because these sets contained predominantly indirect targets. Subsequent analysis of early target genes did show enrichment for CArG boxes, but the number of confirmed direct targets was too small to extract common features of the promoter sequences [21]. To date the most informative dataset to analyse sequences bound in vivo by the MADS complexes has been the global ChIP data for SEP3, mentioned above [25]. The DNA sequences immunoprecipitated with SEP3 showed a clear enrichment for CArG boxes, usually coinciding with the peak of signal intensity for each bound region. The consensus sequence for the bound CArG boxes was relatively flexible, suggesting that SEP3 binds in vivo to a range of variant CArG boxes. The consensus sequences extracted from SEP3 targets in the wild type and in the ag mutant, however, were somewhat different, consistent with the idea that different SEP3containing complexes have preferences for distinct sequences. Furthermore, there was a bias in nucleotide composition flanking the CArG boxes, suggesting that the actual binding site is larger than the core CArG box. Importantly, Kaufmann et al. found that the SEP3-bound regions were also enriched for predicted binding sites of other families of transcription factors, particularly the Gbox (bound by bZIP and bHLH proteins), the TCP-binding sequence and ARF-binding sites. These were also enriched near the peaks of ChIP signals, suggesting that additional transcription factors bind to DNA very close to the SEP3 binding sites. Thus a picture is beginning to emerge of how biases for different CArG boxes and co-operation with other transcription factors binding could resolve the paradox of how in vivo specificity is achieved by organ identity proteins that bind in vitro to similar sequences. A comparable paradox occurs with Hox proteins, which bind to an even more ubiquitous sequence (sharing an -ATTA- core; [7,8]) than the CArG box. It turned out that Hox proteins regulate large numbers of genes in combination with numerous other transcription factors, to the point that they are better considered as “co-factors that modify the activity of other, more specific transcription factors”[40]. The view that organ identity proteins could play a similar role as co-factors for a large array of more specialised transcription factors would also fit the concept that organ identity genes modify all stages of an underlying, leaf-like developmental program.

4. Combinatorial action and the cis-regulatory code Apart from revealing the gene functions required to build floral organs, identifying target genes is important to clarify how different combinations of organ identity proteins select different sets of target promoters. The current model to explain the combina-

5. Conclusion and future challenges The picture that comes into view from all the work described above extends the parallels between floral organ identity genes

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and Hox genes [7,8]. Both types of genes control large sets of downstream genes, which change from early to late stages of development. Their expression is maintained throughout the development of body segments or floral organs by direct auto-regulation, which is a device frequently used to lock gene expression patterns [41]. The sets of known direct targets of both Hox and floral organ identity genes include a disproportionate number of genes with regulatory or signalling roles, although genes with many other functions, including structural functions and metabolism, may also be directly regulated, especially at late stages of development. It is likely that the downstream targets of organ identity genes vary not only with developmental stage, but also between cell types at each stage. Therefore, to fully understand the gene expression network controlled by the organ identity genes, it will be necessary to disentangle overlapping gene expression programs that run in different cells, tissues and stages of organ development. Profiling gene expression in single cell types within complex tissues is possible by purifying protoplasts from tissues in which specific cell types are marked with GFP expression, as shown initially in Arabidopsis roots and more recently in the shoot meristem [42,43]. Given the importance of distinguishing direct from indirect targets, this will need to be complemented by the even greater technical challenge of adapting these methods to perform cell type-specific ChIP. Gene expression and ChIP datasets with temporal and cellular resolution would also help to reveal how the organ identity proteins co-operate with temporally and spatially restricted transcription factors, and how specific promoter sequences serve as a platform for these interactions. Fully understanding the cis-regulatory code would not only explain the spatial patterns of gene expression during floral organ development, but could also reveal how the gene expression programmes are organised temporally. A precedent for this in animal development is provided by work on PHA-4, which is expressed throughout the development of the pharynx in Caenorrhabditis elegans and regulates distinct genes at different stages of pharingeal development. The stage when a target gene was expressed depended in part on whether it contained high- or low-affinity binding sites for PHA-4, with high affinity sites driving earlier expression [44]. Bioinformatics analysis also revealed binding sites of other transcription factors that function co-operatively with the PHA-4 to determine the timing of target gene expression [45]. A complete understanding of how organ identity genes modify the spatial and temporal expression of other genes, however, will require understanding how transcriptional control overlaps with additional levels of regulation. One of these is chromatin accessibility, which might determine which genes are available for regulation by organ identity genes. One example of overlapping control by organ identity genes and chromatin modification is NAP. In addition to being activated by AP3/PI [15], NAP expression also requires methylation of lysine 4 of histone 3, mediated by the Trithorax homologues ATX1 and ATX2 [46]. Transcriptional control can also be filtered through downstream control of RNA processing and stability—a regulatory layer that has just begun to be studied at the global level in flowers [47]. In the long term, all this information will be relevant to simulate and predict the behaviour of the gene expression networks that underpin floral organ development, and the first steps in this direction have already been taken [48]. Quantitative analysis and modelling should also reveal how gene expression is translated into localised changes in cell behaviour and how these add up to produce organs with different shapes and functions. The ultimate goal is to understand how evolutionary changes in gene networks resulted in a dazzling variety of flowers built around a conserved core of regulatory genes.

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