During early stages of reproductive development, individual flower primordia arise in an acropetal spiral from the inflorescence meristem. Undifferentiated ray flower primordia are the first to arise in inflorescences of less than 6 mm in diameter, whereas the first disc flower primordia are detected in inflorescences of approximately 6–8 mm in diameter (Figure 1). We divided early flower development into six different stages that were characterized using scanning electron microscopy (SEM) and histological staining (Figures 1 and 2). Figure 1 demonstrates the first five stages of perfect (fully bisexual) disc flower development. At stage 1, flower primordia are small, undifferentiated bumps, whereas at stage 2, ring-shaped petal primordia begin to form. At stage 3, pappus (whorl 1), petal (whorl 2) and stamen (whorl 3) primordia can be clearly distinguished. Until this point in development, both ray and disc flowers still appear morphologically similar and can only be identified based on their position in the inflorescence (Figure 2A). At stage 4, petals begin to elongate, covering the developing stamen and carpel primordia. Flower organ elongation continues during stages 5 and 6 (Figure 2B). As with SEM, histological analysis reveals no morphological differences between ray and disc flowers at stage 3 (Figure 2C). However, at stage 5, stamens lag behind and are shorter in the female ray flowers compared to the perfect disc flowers (Figures 2B and 2C). Furthermore, at stage 5, the ray flower petals have already fused together, and the shapes of the bilabiate ray flowers with their three highly extended lower lobes plus two minute upper lobes can be distinguished. Conversely, in disc flowers, the developing petals are separated from each other. At stage 6, the differences in petal elongation are even more pronounced. At this stage, pappus bristles are much longer in both flower types, nearly covering the developing primordia (Figures 2A and 2B). In earlier work, we defined the stages of capitulum development based on ray flower petal development 23. Developmental stage 6 in ray flowers and disc flowers as described here corresponds to developmental stages 1 and 3 of the capitulum, respectively 23.

cDNA microarray analysis comparing ray and disc flower primordia was performed separately for developmental stages 3, 5 and 6. The gerbera cDNA microarray contains approximately 9000 probes representing all distinct unigenes from the gerbera EST collection 22. Most of the probes (ca. 80%) printed on the microarray represent transcripts from floral cDNA libraries and genes expressed during inflorescence development 22. Microdissected material for RNA isolation was collected and pooled together from a single clone over a year's time from hundreds of flower primordia across tens of inflorescences. In circumstances such as with gerbera flower primordia sampling, where true biological replicates are not possible due to limited amounts of plant material, pooling is a means to minimize biological variation. For our experiments we also pooled several independent RNA isolations and amplified them prior to dye labelling. Each experiment included four technical replicates each with two dye-swaps. We have verified the present microarray results with real-time reverse transcriptase PCR for 12 random transcripts and for 8 gerbera MADS-box genes (See Additional files 1 and 2).

Following non-linear Lowess normalization of the microarray data, a statistical analysis (one-sample t-test) was performed to discover genes that show significant differences in their expression. The analysis was done separately for each developmental stage. To minimize the number of false positives a Benjamin-Hochberg false discovery rate (FDR) was used to control the Type I family-wise error. Transcripts with p-value < 0.05 were selected as statistically significant. With this analysis, the total numbers of differentially expressed genes were 29 at stage 3, 227 at stage 5, and 2264 at stage 6. Distributions of these genes within and between the flower types are visualized with the Venn-diagram shown in figure 3. Only few expressed genes were shared between stages, demonstrating the rapid transcriptional changes occurring during very early stages of gerbera flower development. The raw data for the experiments presented here have been lodged at the ArrayExpress under accession number E-MEXP-418.

At the earliest developmental stage included in the comparison (3), the disc and ray flowers were morphologically similar. This was reflected at the gene expression level, where few changes are detected. Only 27 transcripts showed significant differences in expression with p-value < 0.05 and fold change more than 1.2 (Table 1). From these, 15 showed greater expression in disc flowers and 12 in ray flowers. In disc flowers most of the genes were related to basic metabolic processes. One of the most abundant transcript upregulated in disc flowers is the gerbera homolog of Vegetative Storage Protein (VSP) gene. VSPs principally accumulate in vacuoles and have a putative role in nitrogen storage and in biotic and abiotic stress responses. In Arabidopsis, VSP mRNAs are highly abundant in flowers, where they may have distinct roles during reproductive development 2425. The gerbera VSP homolog also showed significantly greater expression in stage 6 disc flowers. In ray flowers at stage 3, most of the upregulated genes encoded unknown proteins. However, one gerbera MADS-box gene, GRCD1, showed greater upregulation. GRCD1 has previously been suggested to be required for defining stamen identity in gerbera 1.

The number of differentially expressed genes was found to increase with developmental time, in concordance with increasing morphological differences between the flower types. In disc flowers, the number of genes showing statistically significant changes in expression was 111 at stage 5 and 1156 at stage 6, and in ray flowers, 116 and 1108, respectively. Below, we limit further discussion to the group of genes showing flower-type specific upregulation during both stages 5 and 6 (Table 2). In disc flowers, 49 transcripts were upregulated in both stages 5 and 6 (Table 2). Twenty (41 %) of these gave a BLAST hit while the rest have unknown function.

One of the most significantly upregulated gene had a match to GDSL-lipase. These enzymes are highly abundant in Arabidopsis flowers and are postulated to have specific functions during anther morphogenesis 2526. GDSL-motif lipase genes are also highly expressed throughout tepal development in Iris hollandica 27. Another upregulated gene encoded alpha-expansin, known to be involved in growth and development 2829. The most prominent group of genes, however, were those encoding lipid-transfer proteins, which form a large gene family in gerbera 22. The gerbera C-class MADS-box gene GAGA1 was also more abundantly expressed in disc flowers in comparison to ray flowers at both stages 5 and 6.

42 genes were more strongly expressed in ray flowers than in disc flowers at both stages 5 and 6 (Table 2). Of these, 55 % (23), had unknown functions, and many others encoded ribosomal proteins. Most of the identified genes (36) were also upregulated during early petal development (in ray flowers) in our previous microarray analyses 22 reflecting differences in petal development that occur during early stages of flower-type differentiation.

G0000600014B5 (GEG4), a member of the GASA protein family (Kotilainen, unpublished results), was previously detected in elongating flower scapes in addition to being expressed during early petal development 22. G0000700014E10, annotated as a polyphenol oxidase precursor, was more than 5-fold upregulated in ray flower primordia in comparison to disc flowers. Polyphenol oxidases (PPOs) are ubiquitous in higher plants and are the major cause for tissue browning due to oxidization of phenolic substrates. PPOs have been suggested to have a role in defense against insects and plant pathogens, but they may also be activated by mechanical stress. A PPO homolog encoded by aureusidin synthase has been shown to function in flower coloration 30.

Since transcription factors widely regulate many aspects of development, we examined these genes more closely. In addition to the statistical criteria (p < 0.05) we used a fold change threshold of > 1.2 for differentially expressed transcripts. As described earlier, at stage 3, only the MADS-box gene GRCD1 was detected to have greater expression in ray flowers as compared with the corresponding stage in disc flowers. In both flower types, most of the differentially expressed transcription related genes were expressed during stages 5 and 6, and moreover, they showed stage-specific expression (Tables 3 and 4). Especially prominent was the upregulation of several homeotic MADS-box genes that are known to regulate flower development (discussed separately below).

In addition to MADS-box genes, we identified one gene differentially expressed in disc flowers at stage 5 encoding a putative RING zinc finger protein. RING and variant-RING domain proteins are widespread in plants, and as parts of multiprotein complexes they are involved in diverse cellular functions, e.g., ubiquitination pathways 3132. Two gerbera homeobox HD-ZIP factor genes upregulated in disc flowers shared high sequence similarity with a xylem-specific Zinnia elegans gene 33 as well as with Arabidopsis REVOLUTA (REV) and CORONA (CNA) genes, both members of the HD-ZIP III gene family 3435. HD-ZIP III genes play numerous roles in development, including embryo patterning, vascular development, leaf development (organ polarity) as well as meristem initiation 3536. The gerbera EST database currently includes four clusters of genes that are homologous with Squamosa Promoter Binding Proteins (SBPs) and Squamosa Promoter Binding Like (SPL) proteins. Two of these were upregulated in disc flowers at stage 6. In Arabidopsis, SPL3 has been shown to be involved in floral transition 3738 and SPL8 in pollen sac development 39 but functions for the other 16 SPL genes remain largely unknown. We also identified two genes encoding YABBY transcription factors (G0000800003E02, G0000700008F1). Together with KANADI genes, the YABBY gene family promotes abaxial identity of organs 36. In addition, several transcription factors related to regulation of transcription initiation, chromatin assembly, and mRNA processing were upregulated in disc flowers.

In ray flower primordia, 39 genes were differentially upregulated (Table 4). Many of the transcription factors therein belong to different classes of zinc-finger proteins 40. BLAST searches indicated that G0000100020B08 and G0000600011G06 are homologous with putative flowering-time genes identified in maize (INDETERMINATE1, 41) and Arabidopsis (CONSTANS-like 3). The functional role for the GATA1 zinc-finger-like protein G0000500013B9 is not known as yet. We also identified a LIM domain protein homolog upregulated in ray flowers at stage 6. We have previously observed that pLIM2 22 was more than 8-fold upregulated during late stages of stamen development. Nevertheless, multiple functions have been shown for LIM domain proteins in plants, including transcriptional regulation in the nucleus 424340. Finally, at stage 6, we detected upregulation of the bHLH factor encoded by G0000600012D07, previously isolated as GMYC1 and involved in anthocyanin regulation in gerbera 44.

Several previously characterized gerbera MADS-box genes [4546 Figure 5] showed differential expression during the early stages of ray and disc flower differentiation (Tables 1, 2, 3 and 4). GAGA1 and GAGA2 are C-function genes known to be involved in regulation of stamen and carpel identity in gerbera 47. GDEF1 shares high sequence similarity with GDEF2, another B-function gene in gerbera, but it groups phylogenetically with TM6-like genes of the B-class lineage 48. Its function during gerbera flower development has not yet been established 45. GRCD2 is required for carpel identity, but the gene also controls maintenance of flower meristem identity as well as inflorescence determinacy 49. All of the aforementioned genes are upregulated in bisexual disc flowers in comparison to female ray flowers. PHEP7F1 encodes the SQUAMOSA-like gene GSQUA1 47, and G0000800002C9, also upregulated in disc flowers, encodes a MADS-box gene homologous to Chrysanthemum CDM8, which belongs to the FRUITFULL clade 50.

In ray flowers, GRCD1, which was more expressed already at stage 3, was similarly upregulated at stage 6. In addition to GRCD1, another MADS-box gene, G0000100021A03, showed greater expression in ray flowers at stage 6. G0000100021A3 (including the 3' EST G0000100002C11) is a TM3-like MADS-box gene that showed late petal-specific expression in our previous microarray analyses 22. G0000700003A3 (now named GRCD5), which was significantly more expressed in ray flowers, groups phylogenetically close to the previously identified gerbera gene GRCD1 as well as Arabidopsis SEPALLATA3 (data not shown). GRCD5 shares highest sequence similarity with the CDM44 MADS-box gene from chrysanthemum and FBP2 of petunia 5051. In fact, GRCD5 appears to be orthologous to CDM44 in phylogenetic analysis (data not shown). According to the same phylogenetic analysis (data not shown), G0000200014A7 (GRCD3), which is transcriptionally abundant in ray flowers, lies in the AGL6 clade 52, grouping close together with AGL6, MDMADS11, and ZAG3 and ZAG5 [cf. 1]. G0000200001C06 shows sequence similarity to the chrysanthemum CDM51 and gerbera G0000500017F4 MADS-box genes, the latter of which has a leaf specific expression pattern 22 similar to Arabidopsis AGAMOUS-like MADS-box protein AGL12. We observed similar expression patterns for the gerbera MADS-box genes using real time reverse transcriptase PCR (See Additional file 2).

MADS domain proteins form specific homo- and heterodimers and even higher order complexes to conduct their function 5354. Specific interactions among MADS domain proteins require that they are present in the same cells and tissues under the same developmental stages, and correspondingly, it has been shown that transcripts with overlapping expression patterns are preferred as protein interaction partners 5556. In order to strengthen our observations above and to reveal coordinated expression patterns for gerbera MADS-box genes, we combined our present observations with our previous analyses 22. In an independent analysis we looked for genes that were differentially regulated along with GAGA1 and GRCD1. GAGA1 was chosen because it was differentially expressed in disc flowers at both stages 5 and 6, and GRCD1 due to its early upregulation in ray flowers at stage 3 (Tables 1 and 2). Previously 22, we used the gerbera cDNA microarray to identify inflorescence-specific genes in comparison to leaf tissue as well as genes specific for individual reproductive organs and stages (flower scape, pappus bristles, early and late petal development, and stamens). The samples represented relatively late stages of flower organ development in contrast to the early stages of flower primordium development analyzed here. Standard correlation was used to find transcription factors whose expression correlated (correlation coefficient >0.80) with GAGA1 or GRCD1 across all nine conditions (Figure 4). Table 5 summarizes the identified genes.

The first set of genes represents those that are co-expressed with the C-class gene GAGA1, which was the only MADS-box gene showing significant change in expression in both stages 5 and 6 (Table 2 and 3). Across all nine conditions included in the analysis, 27 genes showed an expression pattern similar to GAGA1. The highest correlation (0.96) was observed for GRCD2, the product of which was previously demonstrated to interact with GAGA1 using yeast two hybrid analysis 49. Furthermore, the second gerbera C-function gene, GAGA2, as well as the B-function genes GGLO1 and GDEF2, were co-expressed with GAGA1. GGLO1, a B-function gene orthologous to Arabidopsis PISTILLATA required for petal and stamen identity 47. also showed disc-flower specific expression at stages 3 and 5 in microarray (when multiple-testing correction was not applied). This result was verified using real time RT-PCR (Table S1b) and northern blotting (data not shown). The functionally unknown TM6-like gene GDEF1, the SEPALLATA-like gene GRCD4, and GSQUA1 were also included among the GAGA1-coexpressed group of genes. In addition to MADS-box genes, several other co-expressed transcription factor genes were identified including those encoding Squamosa Binding Protein Like homolog 3, two zinc finger proteins and an MYB domain factor similar to Antirrhinum MYB305, an early regulator of the phenylpropanoid pathway.

GRCD1, which is already strongly upregulated in ray flowers at stage 3, continuing to stage 6, is co-expressed with the TM3-like MADS-box gene G0000100021A03 in all nine conditions (Figure 4b, Table 5). Two SEPALLATA-like genes also showed a highly correlated expression pattern. Furthermore, we identified a co-expressed gene encoding a MYB domain transcription factor similar to Pisum MYB26 57 a pollen specific LIM domain and a NAM-like protein.

Clear morphological differences between the flower types in stamen and petal development (Figures 2B and 2C) that begin at stage 5 probably reflect differences in cell division and elongation as well as in organ fusion. The most prominent difference is the arrest of stamen development in ray flowers. As also observed in gerbera, a common mechanism for generating unisexual flowers is the selective developmental arrest of either female or male organ primordia, which may occur at different stages of development in different species 58. The most fundamental genes discriminating the sexes in flowering plants are the homeotic B- and C-class genes, which distinguish between carpel and stamen development 58. Based on the ABC model of flower development, stamen identity is determined by activity of both B- and C-class MADS-box genes, while the activity of C-class genes alone controls carpel development 59. However, several studies in eudicot species have failed to provide unambiguous support for a hypothesis that development of unisexual flowers would involve alterations in the expression patterns of the B- or C-function genes, which indicates that the genetic mechanisms in sex determination must act downstream of organ identity 6061626364. In gerbera as well, alterations in B- and C-class MADS-box gene expression are not likely the cause of arrested stamen development in ray flowers. Our results suggest that the role of the SEP-like gene GRCD1, highly upregulated in ray flowers, could be related to the flower-type specific arrest of stamen development. In ray flowers of transgenic plants in which GRCD1 expression was downregulated, sterile staminodes were converted into anthocyanin pigmented petals. However, stamen development was only slightly affected in bisexual disc flowers, where fertile pollen was produced although not released 1. Altogether, these results suggest that the arrest of stamen development is connected to organ identity that is differentially established in ray versus disc flowers. We propose that whorl 3 organ identity establishment involves GRCD1 function, but only in ray flowers.

The microarray data, as supported by real time RT-PCR, suggest that MADS-box proteins may be involved in specific complexes required for the differentiation of individual flower types during inflorescence development in Asteraceae. Differential gene expression of several gerbera MADS-box genes (GGLO1, GDEF2, GAGA1, GAGA2 and GRCD1) had earlier been detected with northern blots representing the same floral developmental stages used in this study (M. Kotilainen, unpublished data). The quantitative differences we observed at the gene expression level may reflect later qualitative protein-protein interaction differences and thereby the composition of specific protein complexes involved in regulation of organ identity in different flower types. Because our expression data revealed genes whose expression is correlated both spatially and temporally in various gerbera tissues, we are able to hypothesize specific protein-protein interactions, some of which have already been confirmed by yeast two-hybrid assays or are supported by transgenic phenotypes. A comprehensive interaction analysis of Arabidopsis MADS-box factors has shown that almost 100% of the interacting proteins have an overlap in expression pattern 56. Coexpression can be taken as an indication for possible in planta interaction 56. Furthermore, we have identified differential expression of new SEP-like genes, which in gerbera and other plants show a diversity of functions beyond those described for Arabidopsis 14965. Figure 5 summarizes our current view of gerbera organ-identity genes, indicating (above the arrow) specific factors that have shown flower-type specific effects in transgenic plants 46.

Upregulation of B- and C-class MADS-box genes as well as the SEP-like gene GRCD2 in central disc flowers probably reflects the major role these genes play in determining normal stamen and carpel identity (carpels only in case of GRCD2). In transgenic plants, suppression of B (GGLO1, GDEF2), C (GAGA1, GAGA2) or GRCD2 function caused similar phenotypes in both flower types 4749. Interestingly, the expression of GSQUA1 and the SEP-like gene GRCD4 correlated with B- and C-class gene expression. Despite its sequence similarity to Arabidopsis APETALA1, GSQUA1 is probably not involved in establishing the A function since its expression was detected at the base of developing flowers, in petals, and in developing vascular bundles of the capitulum 4745. However, GRCD4 was also upregulated during late petal development in our previous microarray studies 22, suggesting that it, together with B and C function genes, may encode a petal-specific SEP gene in gerbera. The function of GDEF1, a TM6-like B-class gene upregulated in disc flowers, is less clear. Unlike GDEF2, which is strongly expressed in both petals and stamens, GDEF1 is most highly expressed in stamens (S. Broholm, unpublished results) indicating function in stamen development. A highly similar gene in petunia (PhTM6) also showed stamen expression and strong protein-protein interaction with the petunia PHGLO2 polypeptide 66. In gerbera, yeast two-hybrid results show that both GDEF1 and GDEF2 form heterodimers with the B function protein GGLO1 (data not shown), whereas neither protein interacts with GRCD2, the function of which is carpel-specific 49. GRCD1 and the TM3-like gene G0000100021A03 are both highly upregulated in ray flowers. This suggests that these two genes may encode interacting proteins involved in the arrest of stamen development. Moreover, the expression of GRCD1 and G0000100021A03 correlated at the flower organ level across all nine conditions analyzed, suggesting that they may have additional functional roles, e.g., during petal development. Specific upregulation phenomena in disc versus ray flowers are summarized in Figure 5 below the arrow, the direction of which follows the capitulum radius.

The capitulum of the Asteraceae has been used historically as a model to study inflorescence meristem development. Inflorescence determination and phyllotaxis have been investigated through wounding experiments, and flower-type specific organ development has been analyzed genetically. Both lines of research have suggested the action of a radial morphogenetic gradient in capitulum development. Cylindrical wounding of early sunflower inflorescences to produce isolated plugs of capitulum receptacle tissue resulted in the development of complete, miniature capitula embedded within larger capitula 6768. Thus, the radially organized sunflower receptacle can be reset into further radially patterned zones via disruption of cell-cell communication. Genetic studies of reproductive characters in Microseris also suggest cell-cell communication radially. The hairy and yellow achene (fruit) traits both show concentric localization in Microseris capitula, although they are independently regulated 69. In the strains examined, hairy achenes (controlled by at least two genes) are always peripheral to smooth achenes, and there is a region of overlap ("half-hairy" achenes) in which individual cells appear to respond specifically to a defining gradient. The yellow-achenes trait also shows radial zonation, the extent of which differs among Microseris strains segregating for two yellow-achenes alleles. Bachmann and colleagues 7069 have hypothesized that the genes responsible for peripheral hairy achenes are participants in the establishment of a radial morphogenetic gradient, and that the alleles of yellow-achenes have different response thresholds for this gradient.

Morphogenetic gradients with distinct patterning effects, such as production of ray and trans versus disc flowers in gerbera, can be set up by simple threshold models of short range activation and long range inhibition, so long as the feedback loops are non-linear 71. An example of a non-linear relationship is an inhibited activator protein that must function as a dimer. MADS domain proteins form specific homo- and heterodimers and even higher-order complexes to conduct their function 5354. The long range (non-cell-autonomous) inhibitor could be a diffusible small molecular weight compound, but it has also been demonstrated that plant transcription factor proteins, including the Antirrhinum MADS domain factors DEF and GLO themselves, can move through plasmodesmata and act non-cell-autonomously 7273. Production of MADS multimers would add significantly to the possible responses to a morphogenetic gradient. In summary, the MADS protein system can provide a simple numerical scaffold (dimers, multimers) upon which to generate great cell, tissue, and organ diversity through differential regulation along a radius. This is well presented in development of the whorled structure of single floral meristems, but by extension, the same general radial gradient mechanisms might operate at the inflorescence level, as suggested by previous work on sunflower and Microseris. Since we have discovered that different MADS domain proteins are differentially expressed in ray versus disc flowers of gerbera (Fig. 5), we hypothesize that developmental control of the Asteraceae capitulum may be more than a simple analog of the single flower that it resembles. Comparative flower/inflorescence research on MADS-box gene transcriptional responses to candidate gradient-establishing molecules, as well as studies of in vivo protein-protein interactions, could help test this hypothesis. If corroborated, it will remain to be demonstrated whether gerbera capitula may have acquired flower-like developmental regulation secondarily, or whether their condensed structure makes a more general pattern easier to detect.

Young inflorescences were fixed, dried in a critical point drying unit and coated with platinum/palladium 49. Samples were examined through a scanning electron microscope (Zeiss DSM 962) in the Electron Microscopy Laboratory of the Institute of Biotechnology at the University of Helsinki.

Young inflorescences were fixed in FAA (50% ethanol, 5% acetic acid, 2% formaldehyde) and transferred through ethanol series into 100% xylene. Samples were embedded in paraffin and cut into 10 μm sections. For histological staining, paraffin was removed with xylene and the sections were stained with saffranin (1% in water) and aniline blue (1% in 50% ethanol). The sections were observed and photographed under a light microscope (Olympus CX41).

Flower primordia of ray and disc flowers were isolated with a scalpel under stereomicroscope and immediately stored in liquid nitrogen. RNA was isolated using Trizol reagent (Invitrogen, Carlsbad CA, USA) with scaled down protocol. At least 10 inflorescences, each bearing hundreds of primordia, were collected and pooled together for RNA isolation in each stage. Each pool included more than one thousand individual flower primordia. Pooling of the samples was used in order to minimize biological variation between samples instead of real biological replicates which were not an option due to the limited amount of material. Each experiment included at least two RNA isolations that were pooled together. Quality and the yield of RNA was analyzed with Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany) using RNA 6000 Nano LabChip^® kit (Agilent Technologies, Waldbronn, Germany) and RNA 6000 ladder (Ambion, Austin, TX, USA).

Microarray experiments followed MIAME standards and can be viewed at the ArrayExpress database. Amplification and labelling of the samples were done using Amino Allyl Message Amp Kit (Ambion, Austin, TX, USA) starting with 2 μg of total RNA. In this total RNA sample several RNA isolations had been pooled together, as described above. Total RNA was isolated from the pools of flower primordia. When more than one amplification was required to get enough amplified RNA (aRNA), several amplifications were done and pooled together. For one hybridization, 7.5 μg of aRNA was further labelled with Cy3 or Cy5 fluorescent dyes (Amersham Biosciences, Little Chalfont Buckinghamshire, UK). For each developmental stage, 4 technical replicates were included from which two were dye-swapped. In practice, several hybridizations were done per each stage and the four technical replicates selected for the analysis were chosen to be of high quality. Prehybridization, hybridization and washes followed previous protocols 22. After hybridization, slides for stages 3 and 5 were scanned with GSI Lumonics ScanArray 5000 (Packard/Perkin Elmer) and for stage 6 using Genetix 5000 AL scanner (Genetix, New Milton, Hampshire, UK) to produce two gray-scale TIFF images. In the GSI Lumonics ScanArray, laser power was changed in order to adjust the two channels whereas in the Genetix 5000, laser value was kept at 100% and PMT gain was changed in order to adjust the two channels. The resolution was 10 μm in all cases.

Genepix pro 5.0 (Axon Instruments, USA) was used to localize the spots and quantify the intensity values in all experiments. The local feature background median was used to subtract the background noise, and visually flawed spots were filtered out from further analysis. Data were normalized and filtered using GeneSpring 7.2 (Silicon Genetics). Due to the non-linearity of the data Lowess normalization was applied. Features with a higher background signal than expression signal were excluded from further analysis. After this the following statistical analysis was performed to find transcripts with significantly altered expression levels between the two samples across four replicates. The data was filtered using one-sample t-test. In order to find genes that were differentially expressed, a t-test was performed for each developmental stage (3, 5 and 6) independently. P-values were adjusted for multiple comparisons by the Benjamini and Hochberg false discovery rate method that controls the Type I family-wise error, and genes having corrected p-value < 0.05 were selected as statistically significant. Here, using one-sample t-test, one can select genes that were differentially expressed between disc and ray flowers, because RNA extracted from both flower types were hybridized on the same chip. Therefore, when the t-test p-value was non-significant, the gene was not differentially expressed, and it was excluded from further analysis. Furthermore, in addition to the p-value criterion a transcript had to show more than 1.2-fold change in order to be considered as differentially expressed.

Three conditions, stage 3, 5 and 6, from the experiments reported here were merged with 6 previously done microarray comparisons 22 to form a combined data set with nine conditions. The previously performed 6 conditions include gene expression analysis of various flower organs (early and late petal development, pappus bristles, stamen and flower scape) that were compared against a pooled reference sample. Comparison of inflorescence sample to leaf sample was also included. These experiments have been described in 22 and the data is available at the ArrayExpress database with accessions E-MEXP-206 and E-MEXP-207. After the data from all nine conditions were merged, the experiment was normalized in GeneSpring 7.2 (Silicon Genetics) using Lowess normalization. Similarity in expression patterns across the various conditions was used as the criterion for co-expression and was calculated using standard correlation in GeneSpring. Mathematically, standard correlation is very similar to Pearson correlation, but it measures the angular separation of expression vectors around zero instead of around one. The transcripts that were functionally annotated to the class 'transcription' were included in the analysis. Transcripts that had a correlation coefficient of more than 0.80 with microarray probes GAGA1 (most highly upregulated in disc flowers) or with GRCD1 (most highly upregulated in ray flowers) were considered to be similarly expressed.

Altogether, 20 transcripts that were printed on the gerbera cDNA microarray were further verified using real time RT-PCR. Total RNA (1.5 μg) was used for cDNA synthesis using TaqMan reverse transcription kit (Applied Biosystems, Roche Molecular Systems Inc., New Jersey, USA) and 5 μl of cDNA was used as a template in PCR reactions. Primers were designed using PrimerExpress software (Applied Biosystems, Foster City, CA, USA). PCR was done in triplicate with 50 nM of primers using ABI 7700 sequence detection systems (Applied Biosystems, Foster City, CA, USA) cycling conditions as default. Ubiquitin was used as a standard against which the raw threshold values (Ct) were normalized in order to get ?Ct values (normalized Ct values). Expression ratio was calculated using the real expression values which in turn were calculated from the formula 2^ΔCt. Each plate included separate ubiquitin standards.

Microarray array design used in this article can be found at the Array-Express http://www.ebi.ac.uk/arrayexpress under accession number A-MEXP-244 and A-MEXP-249 and experiment data under accession E-MEXP-418. Previous microarray results 22, included in the analysis presented in this paper, can be viewed under accessions E-MEXP-206 and E-MEXP-207. EST sequence data from this article can be found in the EMBL/GenBank data libraries under accession numbers AJ750001-AJ766994. EST sequence data and annotations are also available in OpenSputnik database at http://sputnik.btk.fi and at http://www.pgn.cornell.edu.