BACKGROUND
the formation of flowers is one of the main models for studying the molecular mechanisms underlying the control of plant development.
over the past three decades, a large number of regulatory genes, which control a multitude of different processes during flower morphogenesis, have been identified mainly through a combination of forward and reverse genetics approaches .
work in arabidopsis thaliana in particular has led to an understanding of the molecular mechanisms underlying the functions of many of these regulatory genes  <cit> .
furthermore, it has yielded detailed insights into the regulatory hierarchies among genes that play roles in the control of floral organ formation  <cit> .
with the advent of the genomics era, genetic approaches employed to elucidate the regulation of flower development have been complemented by methods such as global transcript profiling and genome-wide localization studies of transcription factor binding sites.
unfortunately, this work has been hampered in arabidopsis by the fact that flowers of this model plant are small and early-stage floral buds are too minute to be dissected reliably through conventional approaches.
also, arabidopsis flowers are initiated sequentially so that all flowers in an inflorescence are at distinct developmental stages  <cit> .
as a consequence, the collection of sufficient numbers of flowers at particular stages for analysis by genomic technologies is challenging especially for early flower development.
to circumvent this problem, a number of approaches have been employed: recently, laser capture microdissection has been used to generate transcriptional profiles of early-stage floral buds  <cit> .
an alternative and largely complementary approach has been the use of floral induction systems, which allow the collection of hundreds of synchronized floral buds from a single plant .
these systems have been employed to study both temporal and spatial gene expression during the early stages of flower development .
other studies have analyzed gene expression in whole inflorescences of wild-type and mutant plants and in some cases relied on the removal of older  buds that may unduly contribute to rna preparations from these tissues .
moreover, transcript profiling was done with wild-type flowers at individual stages and with distinct floral organ types, but this work has been limited to older flowers, as they can be collected with relative ease  <cit> .
specific developmental processes such as male-gametophyte/pollen and female gametophyte/ ovule development have also been studied through transcriptomics experiments, providing detailed information for individual cell and tissue types .
although arabidopsis flower development has been studied extensively over the past ten years through the genomics approaches described above, a continuous series of gene expression from the time of initiation to maturation has been lacking.
obtaining this information could be highly informative as it would provide a comprehensive view of stage-specific gene expression activities over the entire course of development and would constitute an important component of a gene expression map.
furthermore, such a dataset could be used in analyses, in which, for example, data from transcript profiling and genome-wide localization studies are integrated to obtain a better understanding of the gene network that controls flower formation.
in this study, we employed a floral induction system to close this knowledge gap and to monitor temporal gene expression during flower development from the time of initiation to maturation.
we validated the resulting dataset and used it to obtain novel insights into the processes underlying the formation of flowers on a global scale through computational approaches.
RESULTS
temporal gene expression during flower development
to identify patterns of gene expression during flower development from the time of initiation to maturation , we employed a previously described floral induction system, which allows the collection of hundreds of floral buds from a single plant  <cit> .
this system is based on the expression of the floral meristem identity factor apetala <dig>  fused to the hormone-binding domain of the rat glucocorticoid receptor  from the ap <dig> regulatory region  in an ap <dig> cauliflower  double-mutant background.
ap <dig> cal plants accumulate inflorescence-like meristems at their shoot apices  <cit> , and activation of the ap1-gr fusion protein in this background through treatment of the plants with the steroid hormone dexamethasone results in the transformation of these meristems into floral primordia, which subsequently develop in a largely synchronized manner.
however, at intermediate stages, this synchronization is gradually lost likely due to space constraints  <cit> .
despite this overall loss of synchronization, we noticed that flowers at the very tip of the inflorescence heads remained fairly synchronized throughout flower development perhaps due to a larger degree of curvature in his area, which may allow floral buds to develop without coming into contact with neighboring flowers.
for the gene expression profiling experiments, we therefore collected older floral buds  from this region alone, while younger flowers were harvested more liberally from the inflorescences of ap1pro:ap1-gr ap <dig> cal plants .
to obtain expression data for a large number of distinct floral stages, we collected floral buds at  <dig> different time-points either immediately before  or from  <dig> to  <dig> d after the induction of flower development through treatment with dexamethasone .
because early flower development is characterized by dramatic changes in morphology  <cit>  and involves a large number of transcriptional regulators that control important processes such as floral patterning and floral organ specification  <cit> , we collected most samples at those stages with intervals in-between time-points ranging from  <dig>  to  <dig> d. at later stages of development, the intervals for sample collection were extended to  <dig> d .fig.
<dig> analysis of temporal gene expression during flower development.
a-j inflorescences of ap1pro:ap1-gr ap1-1 cal- <dig> plants a before dexamethasone treatment  , and b  <dig> d, c  <dig> d, d  <dig> d, e  <dig> d, f  <dig> d, g  <dig> d, h  <dig> d, i  <dig> d, and j  <dig> d after treatment with a solution containing 10 μm dexamethasone.
the development of flowers on a given inflorescence was largely synchronous until day  <dig>  for later time-points , flowers were harvested from the tip of the inflorescences  after phenotypic assessment.
k experimental set-up used for this study.
floral buds were collected from the inflorescences of ap1pro:ap1-gr ap1-1 cal- <dig> plants at  <dig> time-points immediately before and after treatment with a dexamethasone -containing solution, which induces flower development by activating the ap1-gr fusion protein.
floral buds from the time of initiation until anthesis  were sampled
for microarray analysis of the tissue samples, we employed a common reference design .
we then assessed the resulting data for reproducibility and found that the replicates for the individual time-points correlated well , implying that the progression of flower development and the tissue collection was highly reproducible over the entire course of the experiment.
in order to determine significant expression changes, we applied an f-statistic and searched across the entire dataset for genes with differential expression.
we identified ~ <dig>  genes  that showed differential expression in at least one of fourteen time-points.
because many of these transcriptional changes may be caused by the dramatic alterations in floral size and morphology during the course of development and not by specific gene regulatory events, we next sought to identify genes whose expression changed relatively rapidly.
to this end, we compared gene expression between consecutive as well as near-by  time-points to minimize the effects of morphological alterations and identified  <dig>  genes as differentially expressed .
many of these differentially expressed genes  were detected at intermediate  and late  stages of flower development, and overall, a preponderance of gene activation over repression was observed .
although we found many genes to be repressed immediately after the onset of flower development, this effect was not as pronounced as previously described  <cit> , possibly because of the different floral induction systems and/or different experimental set-ups and data analysis pipelines used.fig.
<dig> expression profiles of known floral regulators.
a-l
m values ) for selected floral regulators  are shown for all time-points.
red, green and blue lines represent data from three biologically independent sets of samples, black lines the mean values of the replicate experiments.
note the high reproducibility of the expression data across all time-points
to validate the results of the microarray experiments, we assessed the expression profiles of genes with known roles in different processes during flower development  and found that they were in concurrence with their published expression patterns.
for example, expression of the floral homeotic genes apetala <dig>  and agamous   strongly increased in early time-points and then remained high throughout most of flower development in agreement with the activation of these genes at stage  <dig> and their continued expression in developing floral organs  <cit> .
down-regulation of the floral repressor short vegetative phase   at early floral stages has been described previously and is dependent on ap <dig> activity  <cit> .
expression of the stem cell regulator clavata <dig>  was high at early stages and then rapidly decreased in intermediate-stage flowers  likely as a consequence of the loss of floral stem cells around stage  <dig> of development  <cit> .
this termination of floral meristems is at least in part due to the activity of knuckles , which we detected to be expressed at intermediate stages , in agreement with its known expression pattern at the base of developing carpels and in stamen primordia  <cit> .
genes with bimodal expression profiles included superman  , which is initially expressed in young floral meristems and at later floral stages during ovule development  <cit> .
strong up-regulation of the regulator of ovule and seed development seedstick  between days  <dig> and  <dig> in our experiment  corresponds to its expression in developing carpels from stage  <dig> onward  <cit> .
duo pollen <dig> , a regulator of male germline development, was found to be expressed in late flower development  in agreement with its specific expression in pollen  <cit> .
aborted microspores , which encodes a master regulator of pollen wall formation, was strongly expressed at intermediate stages and reached a maximum around stages 9- <dig>   as previously described  <cit> .
genes such as nozzle/sporocyteless  , extra microsporocytes1/ extra sporogenous cells  , and dysfunctional tapetum <dig>   were expressed during intermediate stages in agreement with their function in early anther development .
activation of nzz/spl was detected in our experiment around stage  <dig> and thus earlier than what has been reported previously .
this difference might stem from initially low mrna levels, which might hamper a reliable detection in in situ hybridization or reporter gene essays.
we also compared our dataset to those from several previous studies in which temporal  and spatial  <cit>  gene expression during flower development had been analyzed either in early or in late-stage flowers using different floral induction systems, laser capture microdissection of wild-type flowers, or through a comparison of the gene expression profiles of inflorescences of floral mutants and of the wild type, respectively.
for each pair-wise comparison, we found a significant overlap between the datasets and the one described in this study , further validating the results of our time-course experiment.
distribution of functional terms among groups of co-expressed genes
because functionally related genes are often co-expressed during development, we used a k-means algorithm to group the degs into  <dig> clusters with distinct gene expression profiles .
figure  <dig> shows that the majority of degs are predominantly expressed at or after the 9-d time-point.
notable exceptions include genes in clusters  <dig>   <dig> and  <dig>  which are up-regulated during early flower development and are repressed at intermediate to late stages.
also, clusters  <dig> and  <dig> contain genes that are expressed at the earliest floral stages and are subsequently down-regulated.
genes assigned to clusters  <dig> and  <dig> are activated during early flower development when organ primordia are initiated and remain expressed until flowers have reached maturity, suggesting that many of them might play roles during the course of floral organ morphogenesis.fig.
<dig> genes showing differential expression during flower development.
groups of co-expressed genes were identified among  <dig>  differentially expressed genes detected in the time-course experiment.
the heat map shows the results of k-means clustering  used to group genes based on the similarity of their z-scores .
for a different representation of the individual clusters, see figure s3
to obtain insights into the functions of the genes assigned to each of the clusters and to further validate the microarray data, we mapped the groups of co-expressed genes onto an arabidopsis gene expression atlas we had generated previously  <cit>  based on published data .
we then determined the percentage of genes with maximum  and, for comparison, minimum  expression in different groups of related tissue samples.
for some of the clusters, this analysis allowed predictions of the predominant location of gene expression.
for example, a high percentage of genes with maximum expression in pollen was identified in clusters  <dig>   <dig>  8- <dig>  and 13- <dig>  genes assigned to these clusters were predominantly expressed from or after the 9-d time-point and thus at stages when pollen formation occurs  <cit> .
clusters  <dig>   <dig>  and  <dig> contained the highest proportion of genes with maximum expression in meristems, in agreement with the observation that genes in these clusters are strongly expressed during the earliest floral stages, but are repressed towards more intermediate stages when meristematic activity in flowers ceases.
the highest percentage of genes with maximum expression in ovules was found in cluster  <dig>  which contains relatively few genes that are strongly expressed around the  <dig> and 9-day time-points  and thus at the time when ovule development commences  <cit> .fig.
<dig> mapping groups of co-expressed genes onto an arabidopsis gene expression atlas.
a expression data for an arabidopsis gene expression atlas were obtained for genes assigned to each of the 15 k-means clusters and hierarchical clustering was performed.
results for cluster  <dig> are shown as an example.
individual tissue and organ samples of the gene expression atlas  were grouped together as indicated.
note a preponderance of expression in stamen and pollen samples.
b and c the number of genes in each cluster with b maximum and c minimum expression in each of the tissue samples  is shown
we also subjected the groups of co-expressed genes to a gene ontology  analysis to identify functionally related genes that are significantly enriched  in the individual clusters .
go terms directly associated with flower formation  and/or floral meristem development  were found to be enriched, in particular, in clusters  <dig> and  <dig>  as well as in clusters  <dig> and  <dig> .
as described above, these clusters contain genes that are repressed at early to mid-stages  or are activated during early flower development  and remain expressed at least until the end of the intermediate phase of flower development.
in agreement with the over-representation of flower-related go terms in these clusters, they contain many of the regulatory genes  known to be involved in controlling the early phase of flower development .
genes associated with the term ‘pollen development’ were enriched in clusters  <dig> and  <dig>  which contain genes with maximal expression around day  <dig> of the experiment and hence at a time  when the microspore mother cells appear and meiosis takes place  <cit> .
genes involved in cell differentiation were enriched in clusters  <dig> and  <dig>  which contain genes with predominant expression at late stages of flower development .
many of these genes exhibit maximum expression in pollen  and thus, may be involved to a large extent in the differentiation of microspores into pollen grains.
genes involved in the response to different phytohormones such as jasmonic acid, auxin, and abscisic acid were detected as enriched predominantly in cluster  <dig>  in agreement with the known roles of these hormones in various processes during late-stage flower development, which include stamen and pollen formation as well as the maturation of petals  <cit> .
in contrast, genes involved in the response to gibberellin were over-represented in cluster  <dig>  which contains genes that are induced at the end of the early phase of flower development and remain active until floral maturity has been reached.
in agreement with this observation, it has been shown that gibberellins are required for proper floral organ growth and elongation  <cit> .
in sum, the results of these analyses allowed us to attribute specific functions to the individual clusters that together account for many of the processes known to occur during flower development.fig.
<dig> gene ontology terms enriched in the dataset.
adjusted p-values for selected go terms related to a developmental functions and b cellular and regulatory processes are indicated for each cluster through color-coding .
for a full list of go terms enriched in the dataset, see additional file 5
distribution of target genes of floral organ identity factors
floral organ identity factors are necessary and sufficient for the specification and development of the different types of floral organs  <cit> .
they act in a combinatorial manner as predicted by the well-supported bce model of floral organ identity specification .
insights into the functions of these master regulators, which  all belong to the family of mads-domain proteins and are components of higher-order regulatory protein complexes  <cit> , have been obtained in recent years through a combination of genome-wide localization studies and gene perturbation experiments  <cit> .
this work has resulted in the identification of some of their direct target genes and of the cellular and developmental processes they control.
furthermore, it has been shown that the floral organ identity factors bind to many of the same sites in the arabidopsis genome  <cit>  and that their global binding patterns undergo changes as flower development progresses, at least in part as a consequence of stage-specific alterations in chromatin accessibility  <cit> .
also, the majority of genes bound by these transcription factors at early floral stages do not respond transcriptionally when the activities of the floral homeotic genes are perturbed  <cit> .
while the molecular mechanisms underlying these observations are currently not well understood, it is clear that from binding data alone it is difficult to identify their bona fide target genes.
to test whether we could find evidence for the differential expression of genes that are bound by the floral organ identity factors, we projected the global binding patterns of ap <dig>  sepallata <dig> , ap <dig>  pistillata , and ag onto the dataset from the flowering time-course experiment .
specifically, we identified the percentage of genes in each of the  <dig> clusters of co-expressed genes that contain binding sites for these transcription factors in their putative regulatory regions .
while binding data for ap <dig>  pi and ag are currently available only for ~ stage  <dig> flowers  <cit> , for ap <dig> and sep <dig>  binding data for three distinct stages  have been generated  <cit> .
largely independent of the transcription factor under study, we found the highest degree of binding site enrichment in clusters  <dig>   <dig>   <dig>  and  <dig> .
cluster  <dig> also showed a significant enrichment for genes with binding sites, but only for sep <dig> and ap <dig>  and not at the earliest  time-point.
the genes assigned to these different clusters have in common that their transcription changes at the time or shortly after the expression of the floral organ identity genes commences around stage  <dig>  furthermore, they contain many genes associated with the specification of floral organ identity, as well as the regulation of floral organ development and meristem determinacy  and thus processes that are known to be under control of the floral organ identity factors  <cit> .
hence, genes in these clusters containing binding sites for the mads-domain proteins are good candidates for target genes.
in fact, they do contain many of the genes known to act directly downstream of these floral regulators .
however, one caveat of this analysis is that the floral organ identity factors appear to have largely distinct sets of target genes despite their overlapping binding patterns  <cit> .
therefore, while genes that are differentially expressed during early flower development and that contain binding sites for mads-domain proteins are likely under control of floral organ identity factors, the exact regulatory complex that might be active in the regulation of a given gene cannot be readily deduced without additional data from floral organ identity gene-specific perturbation experiments.fig.
<dig> distribution of genes with binding sites for floral organ identity factors.
the percentage of genes in each cluster bound by a sep <dig>  b ap <dig>  and c ap <dig>  pi, and ag, respectively, is shown.
for a and b, binding data for sep <dig> and ap <dig>  respectively, at three different time points after ap1-gr activation were used for analysis:  <dig> d ,  <dig> d , and  <dig> d .
for c, binding data for ap <dig> , pi , and ag   <dig> d after ap1-gr activation were used.
in all panels, bars without error bars show the results of the comparisons between binding data for the individual transcription factors and the clusters of co-expressed genes, while bars with error bars show the mean percentage of genes bound by a given floral homeotic transcription factor at the indicated time-point in equally sized groups of genes randomly selected from the dataset of  <dig>  degs.
error bars indicate one standard deviation calculated based on the results of  <dig> iterations
in addition to clusters with binding site enrichments, we also found clusters that are significantly depleted for binding sites of the floral organ identity factors.
these included especially clusters  <dig>   <dig>  and  <dig>  which contain genes with predominant expression in the time-course experiment at  <dig>   <dig>  and  <dig> d, respectively .
as described above, these clusters comprise in all probability many genes involved in microsporogenesis and pollen development, a process that can progress without the direct involvement of the floral organ identity factors  <cit> .
taken together, this analysis shows that the results of our transcriptomics study can be used as a reference to integrate different genome-wide datasets and to identify candidates for transcription factor target genes.
distribution of paralogs within groups of co-expressed genes
in plants, duplicated genes that are retained in a genome are often functionally redundant, although sub- or neofunctionalization may lead to paralogous genes that have only partially overlapping activities or that are employed in entirely different developmental processes, respectively  <cit> .
shared activities of paralogous genes typically go along with overlapping expression patterns.
therefore, one would expect to find in the clusters of co-expressed genes that paralogs are enriched relative to their genome-wide distribution.
in fact, it has been shown previously that paralogous genes are over-represented in some but not all groups of genes with predominant expression at certain stages of early flower development  <cit> .
to test whether this unequal distribution of paralogs extends to intermediate or late stages of flower development, we determined paralogs in each of the  <dig> clusters described in fig.
<dig> .
as expected, we found that the percentage of paralogs was significantly  increased in all clusters relative to their genome-wide distribution and to a lesser extent  relative to their distribution within the  <dig>  degs as well .
notably, the enrichment of paralogs within the clusters varied considerably, with clusters  <dig>  11- <dig>  and  <dig> having the highest enrichment values .
in agreement with the idea that genes involved in floral organ development exhibit an increased level of genetic redundancy  <cit> , the genes in these clusters have in common that they are activated during early or intermediate  stages of flower development and many of them have known functions in floral organ morphogenesis and in the control of floral meristem determinacy .
in sum, our results further highlight the varying degree to which paralogous genes contribute to different processes during flower development.
whether such an unequal distribution of paralogs among groups of co-expressed genes extends to other processes during plant development is currently unknown.fig.
<dig> distribution of paralogs in groups of co-expressed genes.
the percentage of paralogs in each cluster of co-expressed genes  was determined as described in methods.
to identify clusters with a significant enrichment of paralogous genes, the mean percentage of paralogs was determined in equally sized groups of genes randomly selected from the dataset of  <dig>  degs  and from the arabidopsis genome , respectively.
error bars indicate one standard deviation calculated based on the results of  <dig> iterations
CONCLUSIONS
the results of our transcriptomics analysis of flower development, which covered most stages from the time of initiation until maturation, shows that the formation of flowers involves the differential expression of at least a quarter of the genes in the arabidopsis genome.
while many gene expression changes occur late in development and are likely due to the activation of specific gene sets in developing pollen and - to a lesser extent - ovules, genes with regulatory functions often exhibit intermittent expression during early and late floral stages.
through computational analyses, we have been able to assign functions to groups of co-expressed genes and to provide temporal information on when these processes likely occur during the almost two weeks during which flowers develop from a small number of meristematic cells into a highly complex structure with different organs, tissues and cell types.
using binding data for selected floral organ identity factors, we have further demonstrated that the results of our transcriptomics experiment can help to interpret and mine datasets from genome-wide localization studies.
our data also provide an important component of a gene expression map for flower development.
through the use of techniques such as translating ribosome affinity purification   <cit>  or isolation of nuclei tagged in specific cell types   <cit> , it should be possible to extend this map by introducing detailed spatial information on gene expression for all floral stages.

 the formation of flowers is one of the main models for studying the molecular mechanisms underlying the control of plant development. over the past three decades, a large number of regulatory genes, which control a multitude of different processes during flower morphogenesis, have been identified mainly through a combination of forward and reverse genetics approaches. work in arabidopsis thaliana in particular has led to an understanding of the molecular mechanisms underlying the functions of many of these regulatory genes. furthermore, it has yielded detailed insights into the regulatory hierarchies among genes that play roles in the control of floral organ formation. with the advent of the genomics era, genetic approaches employed to elucidate the regulation of flower development have been complemented by methods such as global transcript profiling and genome-wide localization studies of transcription factor binding sites. unfortunately, this work has been hampered in arabidopsis by the fact that flowers of this model plant are small and early-stage floral buds are too minute to be dissected reliably through conventional approaches. also, arabidopsis flowers are initiated sequentially so that all flowers in an inflorescence are at distinct developmental stages. as a consequence, the collection of sufficient numbers of flowers at particular stages for analysis by genomic technologies is challenging especially for early flower development. to circumvent this problem, a number of approaches have been employed: recently, laser capture microdissection has been used to generate transcriptional profiles of early-stage floral buds. an alternative and largely complementary approach has been the use of floral induction systems, which allow the collection of hundreds of synchronized floral buds from a single plant. these systems have been employed to study both temporal and spatial gene expression during the early stages of flower development. other studies have analyzed gene expression in whole inflorescences of wild-type and mutant plants and in some cases relied on the removal of older buds that may unduly contribute to rna preparations from these tissues. moreover, transcript profiling was done with wild-type flowers at individual stages and with distinct floral organ types, but this work has been limited to older flowers, as they can be collected with relative ease. specific developmental processes such as male-gametophyte/pollen and female gametophyte/ ovule development have also been studied through transcriptomics experiments, providing detailed information for individual cell and tissue types. although arabidopsis flower development has been studied extensively over the past ten years through the genomics approaches described above, a continuous series of gene expression from the time of initiation to maturation has been lacking. obtaining this information could be highly informative as it would provide a comprehensive view of stage-specific gene expression activities over the entire course of development and would constitute an important component of a gene expression map. furthermore, such a dataset could be used in analyses, in which, for example, data from transcript profiling and genome-wide localization studies are integrated to obtain a better understanding of the gene network that controls flower formation. in this study, we employed a floral induction system to close this knowledge gap and to monitor temporal gene expression during flower development from the time of initiation to maturation. we validated the resulting dataset and used it to obtain novel insights into the processes underlying the formation of flowers on a global scale through computational approaches.  temporal gene expression during flower development to identify patterns of gene expression during flower development from the time of initiation to maturation, we employed a previously described floral induction system, which allows the collection of hundreds of floral buds from a single plant. this system is based on the expression of the floral meristem identity factor apetala fused to the hormone-binding domain of the rat glucocorticoid receptor from the ap regulatory region in an ap cauliflower double-mutant background. ap cal plants accumulate inflorescence-like meristems at their shoot apices, and activation of the ap1-gr fusion protein in this background through treatment of the plants with the steroid hormone dexamethasone results in the transformation of these meristems into floral primordia, which subsequently develop in a largely synchronized manner. however, at intermediate stages, this synchronization is gradually lost likely due to space constraints. despite this overall loss of synchronization, we noticed that flowers at the very tip of the inflorescence heads remained fairly synchronized throughout flower development perhaps due to a larger degree of curvature in his area, which may allow floral buds to develop without coming into contact with neighboring flowers. for the gene expression profiling experiments, we therefore collected older floral buds from this region alone, while younger flowers were harvested more liberally from the inflorescences of ap1pro:ap1-gr ap cal plants. to obtain expression data for a large number of distinct floral stages, we collected floral buds at different time-points either immediately before or from to d after the induction of flower development through treatment with dexamethasone. because early flower development is characterized by dramatic changes in morphology and involves a large number of transcriptional regulators that control important processes such as floral patterning and floral organ specification, we collected most samples at those stages with intervals in-between time-points ranging from to d. at later stages of development, the intervals for sample collection were extended to d.fig. analysis of temporal gene expression during flower development. a-j inflorescences of ap1pro:ap1-gr ap1-1 cal- plants a before dexamethasone treatment, and b d, c d, d d, e d, f d, g d, h d, i d, and j d after treatment with a solution containing 10 μm dexamethasone. the development of flowers on a given inflorescence was largely synchronous until day for later time-points, flowers were harvested from the tip of the inflorescences after phenotypic assessment. k experimental set-up used for this study. floral buds were collected from the inflorescences of ap1pro:ap1-gr ap1-1 cal- plants at time-points immediately before and after treatment with a dexamethasone -containing solution, which induces flower development by activating the ap1-gr fusion protein. floral buds from the time of initiation until anthesis were sampled for microarray analysis of the tissue samples, we employed a common reference design. we then assessed the resulting data for reproducibility and found that the replicates for the individual time-points correlated well, implying that the progression of flower development and the tissue collection was highly reproducible over the entire course of the experiment. in order to determine significant expression changes, we applied an f-statistic and searched across the entire dataset for genes with differential expression. we identified ~ genes that showed differential expression in at least one of fourteen time-points. because many of these transcriptional changes may be caused by the dramatic alterations in floral size and morphology during the course of development and not by specific gene regulatory events, we next sought to identify genes whose expression changed relatively rapidly. to this end, we compared gene expression between consecutive as well as near-by time-points to minimize the effects of morphological alterations and identified genes as differentially expressed. many of these differentially expressed genes were detected at intermediate and late stages of flower development, and overall, a preponderance of gene activation over repression was observed. although we found many genes to be repressed immediately after the onset of flower development, this effect was not as pronounced as previously described, possibly because of the different floral induction systems and/or different experimental set-ups and data analysis pipelines used.fig. expression profiles of known floral regulators. a-l m values ) for selected floral regulators are shown for all time-points. red, green and blue lines represent data from three biologically independent sets of samples, black lines the mean values of the replicate experiments. note the high reproducibility of the expression data across all time-points to validate the results of the microarray experiments, we assessed the expression profiles of genes with known roles in different processes during flower development and found that they were in concurrence with their published expression patterns. for example, expression of the floral homeotic genes apetala and agamous strongly increased in early time-points and then remained high throughout most of flower development in agreement with the activation of these genes at stage and their continued expression in developing floral organs. down-regulation of the floral repressor short vegetative phase at early floral stages has been described previously and is dependent on ap activity. expression of the stem cell regulator clavata was high at early stages and then rapidly decreased in intermediate-stage flowers likely as a consequence of the loss of floral stem cells around stage of development. this termination of floral meristems is at least in part due to the activity of knuckles, which we detected to be expressed at intermediate stages, in agreement with its known expression pattern at the base of developing carpels and in stamen primordia. genes with bimodal expression profiles included superman, which is initially expressed in young floral meristems and at later floral stages during ovule development. strong up-regulation of the regulator of ovule and seed development seedstick between days and in our experiment corresponds to its expression in developing carpels from stage onward. duo pollen, a regulator of male germline development, was found to be expressed in late flower development in agreement with its specific expression in pollen. aborted microspores, which encodes a master regulator of pollen wall formation, was strongly expressed at intermediate stages and reached a maximum around stages 9- as previously described. genes such as nozzle/sporocyteless, extra microsporocytes1/ extra sporogenous cells, and dysfunctional tapetum were expressed during intermediate stages in agreement with their function in early anther development. activation of nzz/spl was detected in our experiment around stage and thus earlier than what has been reported previously. this difference might stem from initially low mrna levels, which might hamper a reliable detection in in situ hybridization or reporter gene essays. we also compared our dataset to those from several previous studies in which temporal and spatial gene expression during flower development had been analyzed either in early or in late-stage flowers using different floral induction systems, laser capture microdissection of wild-type flowers, or through a comparison of the gene expression profiles of inflorescences of floral mutants and of the wild type, respectively. for each pair-wise comparison, we found a significant overlap between the datasets and the one described in this study, further validating the results of our time-course experiment. distribution of functional terms among groups of co-expressed genes because functionally related genes are often co-expressed during development, we used a k-means algorithm to group the degs into clusters with distinct gene expression profiles. figure  shows that the majority of degs are predominantly expressed at or after the 9-d time-point. notable exceptions include genes in clusters and which are up-regulated during early flower development and are repressed at intermediate to late stages. also, clusters and contain genes that are expressed at the earliest floral stages and are subsequently down-regulated. genes assigned to clusters and are activated during early flower development when organ primordia are initiated and remain expressed until flowers have reached maturity, suggesting that many of them might play roles during the course of floral organ morphogenesis.fig. genes showing differential expression during flower development. groups of co-expressed genes were identified among differentially expressed genes detected in the time-course experiment. the heat map shows the results of k-means clustering used to group genes based on the similarity of their z-scores. for a different representation of the individual clusters, see figure s3 to obtain insights into the functions of the genes assigned to each of the clusters and to further validate the microarray data, we mapped the groups of co-expressed genes onto an arabidopsis gene expression atlas we had generated previously based on published data. we then determined the percentage of genes with maximum and, for comparison, minimum expression in different groups of related tissue samples. for some of the clusters, this analysis allowed predictions of the predominant location of gene expression. for example, a high percentage of genes with maximum expression in pollen was identified in clusters 8- and 13- genes assigned to these clusters were predominantly expressed from or after the 9-d time-point and thus at stages when pollen formation occurs. clusters and contained the highest proportion of genes with maximum expression in meristems, in agreement with the observation that genes in these clusters are strongly expressed during the earliest floral stages, but are repressed towards more intermediate stages when meristematic activity in flowers ceases. the highest percentage of genes with maximum expression in ovules was found in cluster which contains relatively few genes that are strongly expressed around the and 9-day time-points and thus at the time when ovule development commences.fig. mapping groups of co-expressed genes onto an arabidopsis gene expression atlas. a expression data for an arabidopsis gene expression atlas were obtained for genes assigned to each of the 15 k-means clusters and hierarchical clustering was performed. results for cluster are shown as an example. individual tissue and organ samples of the gene expression atlas were grouped together as indicated. note a preponderance of expression in stamen and pollen samples. b and c the number of genes in each cluster with b maximum and c minimum expression in each of the tissue samples is shown we also subjected the groups of co-expressed genes to a gene ontology analysis to identify functionally related genes that are significantly enriched in the individual clusters. go terms directly associated with flower formation and/or floral meristem development were found to be enriched, in particular, in clusters and as well as in clusters and. as described above, these clusters contain genes that are repressed at early to mid-stages or are activated during early flower development and remain expressed at least until the end of the intermediate phase of flower development. in agreement with the over-representation of flower-related go terms in these clusters, they contain many of the regulatory genes known to be involved in controlling the early phase of flower development. genes associated with the term ‘pollen development’ were enriched in clusters and which contain genes with maximal expression around day of the experiment and hence at a time when the microspore mother cells appear and meiosis takes place. genes involved in cell differentiation were enriched in clusters and which contain genes with predominant expression at late stages of flower development. many of these genes exhibit maximum expression in pollen and thus, may be involved to a large extent in the differentiation of microspores into pollen grains. genes involved in the response to different phytohormones such as jasmonic acid, auxin, and abscisic acid were detected as enriched predominantly in cluster in agreement with the known roles of these hormones in various processes during late-stage flower development, which include stamen and pollen formation as well as the maturation of petals. in contrast, genes involved in the response to gibberellin were over-represented in cluster which contains genes that are induced at the end of the early phase of flower development and remain active until floral maturity has been reached. in agreement with this observation, it has been shown that gibberellins are required for proper floral organ growth and elongation. in sum, the results of these analyses allowed us to attribute specific functions to the individual clusters that together account for many of the processes known to occur during flower development.fig. gene ontology terms enriched in the dataset. adjusted p-values for selected go terms related to a developmental functions and b cellular and regulatory processes are indicated for each cluster through color-coding. for a full list of go terms enriched in the dataset, see additional file 5 distribution of target genes of floral organ identity factors floral organ identity factors are necessary and sufficient for the specification and development of the different types of floral organs. they act in a combinatorial manner as predicted by the well-supported bce model of floral organ identity specification. insights into the functions of these master regulators, which all belong to the family of mads-domain proteins and are components of higher-order regulatory protein complexes, have been obtained in recent years through a combination of genome-wide localization studies and gene perturbation experiments. this work has resulted in the identification of some of their direct target genes and of the cellular and developmental processes they control. furthermore, it has been shown that the floral organ identity factors bind to many of the same sites in the arabidopsis genome and that their global binding patterns undergo changes as flower development progresses, at least in part as a consequence of stage-specific alterations in chromatin accessibility. also, the majority of genes bound by these transcription factors at early floral stages do not respond transcriptionally when the activities of the floral homeotic genes are perturbed. while the molecular mechanisms underlying these observations are currently not well understood, it is clear that from binding data alone it is difficult to identify their bona fide target genes. to test whether we could find evidence for the differential expression of genes that are bound by the floral organ identity factors, we projected the global binding patterns of ap sepallata, ap pistillata, and ag onto the dataset from the flowering time-course experiment. specifically, we identified the percentage of genes in each of the clusters of co-expressed genes that contain binding sites for these transcription factors in their putative regulatory regions. while binding data for ap pi and ag are currently available only for ~ stage flowers, for ap and sep binding data for three distinct stages have been generated. largely independent of the transcription factor under study, we found the highest degree of binding site enrichment in clusters and. cluster also showed a significant enrichment for genes with binding sites, but only for sep and ap and not at the earliest time-point. the genes assigned to these different clusters have in common that their transcription changes at the time or shortly after the expression of the floral organ identity genes commences around stage furthermore, they contain many genes associated with the specification of floral organ identity, as well as the regulation of floral organ development and meristem determinacy and thus processes that are known to be under control of the floral organ identity factors. hence, genes in these clusters containing binding sites for the mads-domain proteins are good candidates for target genes. in fact, they do contain many of the genes known to act directly downstream of these floral regulators. however, one caveat of this analysis is that the floral organ identity factors appear to have largely distinct sets of target genes despite their overlapping binding patterns. therefore, while genes that are differentially expressed during early flower development and that contain binding sites for mads-domain proteins are likely under control of floral organ identity factors, the exact regulatory complex that might be active in the regulation of a given gene cannot be readily deduced without additional data from floral organ identity gene-specific perturbation experiments.fig. distribution of genes with binding sites for floral organ identity factors. the percentage of genes in each cluster bound by a sep b ap and c ap pi, and ag, respectively, is shown. for a and b, binding data for sep and ap respectively, at three different time points after ap1-gr activation were used for analysis: d, d, and d. for c, binding data for ap, pi, and ag d after ap1-gr activation were used. in all panels, bars without error bars show the results of the comparisons between binding data for the individual transcription factors and the clusters of co-expressed genes, while bars with error bars show the mean percentage of genes bound by a given floral homeotic transcription factor at the indicated time-point in equally sized groups of genes randomly selected from the dataset of degs. error bars indicate one standard deviation calculated based on the results of iterations in addition to clusters with binding site enrichments, we also found clusters that are significantly depleted for binding sites of the floral organ identity factors. these included especially clusters and which contain genes with predominant expression in the time-course experiment at and d, respectively. as described above, these clusters comprise in all probability many genes involved in microsporogenesis and pollen development, a process that can progress without the direct involvement of the floral organ identity factors. taken together, this analysis shows that the results of our transcriptomics study can be used as a reference to integrate different genome-wide datasets and to identify candidates for transcription factor target genes. distribution of paralogs within groups of co-expressed genes in plants, duplicated genes that are retained in a genome are often functionally redundant, although sub- or neofunctionalization may lead to paralogous genes that have only partially overlapping activities or that are employed in entirely different developmental processes, respectively. shared activities of paralogous genes typically go along with overlapping expression patterns. therefore, one would expect to find in the clusters of co-expressed genes that paralogs are enriched relative to their genome-wide distribution. in fact, it has been shown previously that paralogous genes are over-represented in some but not all groups of genes with predominant expression at certain stages of early flower development. to test whether this unequal distribution of paralogs extends to intermediate or late stages of flower development, we determined paralogs in each of the clusters described in fig. . as expected, we found that the percentage of paralogs was significantly increased in all clusters relative to their genome-wide distribution and to a lesser extent relative to their distribution within the degs as well. notably, the enrichment of paralogs within the clusters varied considerably, with clusters 11- and having the highest enrichment values. in agreement with the idea that genes involved in floral organ development exhibit an increased level of genetic redundancy, the genes in these clusters have in common that they are activated during early or intermediate stages of flower development and many of them have known functions in floral organ morphogenesis and in the control of floral meristem determinacy. in sum, our results further highlight the varying degree to which paralogous genes contribute to different processes during flower development. whether such an unequal distribution of paralogs among groups of co-expressed genes extends to other processes during plant development is currently unknown.fig. distribution of paralogs in groups of co-expressed genes. the percentage of paralogs in each cluster of co-expressed genes was determined as described in methods. to identify clusters with a significant enrichment of paralogous genes, the mean percentage of paralogs was determined in equally sized groups of genes randomly selected from the dataset of degs and from the arabidopsis genome, respectively. error bars indicate one standard deviation calculated based on the results of iterations  the results of our transcriptomics analysis of flower development, which covered most stages from the time of initiation until maturation, shows that the formation of flowers involves the differential expression of at least a quarter of the genes in the arabidopsis genome. while many gene expression changes occur late in development and are likely due to the activation of specific gene sets in developing pollen and - to a lesser extent - ovules, genes with regulatory functions often exhibit intermittent expression during early and late floral stages. through computational analyses, we have been able to assign functions to groups of co-expressed genes and to provide temporal information on when these processes likely occur during the almost two weeks during which flowers develop from a small number of meristematic cells into a highly complex structure with different organs, tissues and cell types. using binding data for selected floral organ identity factors, we have further demonstrated that the results of our transcriptomics experiment can help to interpret and mine datasets from genome-wide localization studies. our data also provide an important component of a gene expression map for flower development. through the use of techniques such as translating ribosome affinity purification or isolation of nuclei tagged in specific cell types, it should be possible to extend this map by introducing detailed spatial information on gene expression for all floral stages. 
we analyzed groups of co-expressed genes for over-represented cellular and developmental functions through gene ontology analysis and found that they could be assigned specific patterns of activities, which are in agreement with the progression of flower development. these genes comprise many known floral regulators and we found that the expression profiles for these regulators match their known expression patterns, thus validating the dataset. here, we used a floral induction system to close this information gap and to generate a reference dataset for stage-specific gene expression during flower formation. using a floral induction system, we collected floral buds at different stages from the time of initiation until maturation. our results highlight and describe the dynamic expression changes undergone by a large number of genes during flower development. they further provide a comprehensive reference dataset for temporal gene expression during flower formation and we demonstrate that it can be used to integrate data from other genomics approaches such as genome-wide localization studies of transcription factor binding sites. although several studies have explored gene expression during flower development in the model plant arabidopsis thaliana on a genome-wide scale, a continuous series of expression data from the earliest floral stages until maturation has been lacking. using whole-genome microarray analysis, we identified genes that exhibit rapid expression changes during flower development. we further found that the distribution of paralogs among groups of co-expressed genes varies considerably, with genes expressed predominantly at early and intermediate stages of flower development showing the highest proportion of such genes. arabidopsis thalianaflower developmentorgan specificationtranscriptomicstemporal gene expressionparaloggene expression atlasissue-copyright-statement© the author 2015 the formation of flowers is one of the main model systems to elucidate the molecular mechanisms that control developmental processes in plants. furthermore, by mapping binding sites of floral organ identity factors onto our dataset, we were able to identify gene groups that are likely predominantly under control of these transcriptional regulators.
 the formation of flowers is one of the main model systems to elucidate the molecular mechanisms that control developmental processes in plants. although several studies have explored gene expression during flower development in the model plant arabidopsis thaliana on a genome-wide scale, a continuous series of expression data from the earliest floral stages until maturation has been lacking. here, we used a floral induction system to close this information gap and to generate a reference dataset for stage-specific gene expression during flower formation.  using a floral induction system, we collected floral buds at different stages from the time of initiation until maturation. using whole-genome microarray analysis, we identified genes that exhibit rapid expression changes during flower development. these genes comprise many known floral regulators and we found that the expression profiles for these regulators match their known expression patterns, thus validating the dataset. we analyzed groups of co-expressed genes for over-represented cellular and developmental functions through gene ontology analysis and found that they could be assigned specific patterns of activities, which are in agreement with the progression of flower development. furthermore, by mapping binding sites of floral organ identity factors onto our dataset, we were able to identify gene groups that are likely predominantly under control of these transcriptional regulators. we further found that the distribution of paralogs among groups of co-expressed genes varies considerably, with genes expressed predominantly at early and intermediate stages of flower development showing the highest proportion of such genes.  our results highlight and describe the dynamic expression changes undergone by a large number of genes during flower development. they further provide a comprehensive reference dataset for temporal gene expression during flower formation and we demonstrate that it can be used to integrate data from other genomics approaches such as genome-wide localization studies of transcription factor binding sites. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords arabidopsis thalianaflower developmentorgan specificationtranscriptomicstemporal gene expressionparaloggene expression atlasissue-copyright-statement© the author 2015 