BACKGROUND
in the co-evolution between plants and insects, the development of floral monosymmetry was an important step, facilitating angiosperm speciation as a response to the adaptation to the visual senses of insect pollinators  <cit> .
monosymmetry, thus, likely functioned as a morphological key innovation and evolved independently in different angiosperm lineages  <cit> .
groundbreaking research almost two decades ago identified the tcp transcription factor cycloidea  as the molecular key regulator of monosymmetry development in antirrhinum  <cit> .
cyc and its paralog dichotoma are expressed in the adaxial part of developing antirrhinum flowers, where they guide the acquisition of adaxial identities of second and third whorl organs  <cit> .
cyc belongs to the cyc <dig> clade of the tcp transcription factor family  <cit>  and in all core eudicot species analysed so far, monosymmetry development is controlled by cyc <dig> clade genes .
the majority of crucifers  develop a polysymmetric corolla and only six genera form flowers with two petal pairs of different sizes  <cit> .
in iberis amara, unequal petal pair formation correlates with a stronger expression of the cyc <dig> clade gene iatcp <dig> in the smaller, adaxial petals.
comparison of adaxial and abaxial epidermal cell sizes revealed that petal size differences are due to a differential rate of cell proliferation  <cit> .
in a peloric flower variant, forming only large, abaxialized petals, the iatcp <dig> expression is dramatically decreased.
transgenic arabidopsis plants overexpressing the cruciferous cyc <dig> transcription factors iatcp <dig> from iberis amara and tcp <dig> from arabidopsis, both, produce similar flowers with smaller petals.
for plants overexpressing iatcp <dig> this was shown to be due to a reduction in cell number  <cit> .
contrarily, ectopic expression of cyc from antirrhinum results in transgenic plants forming flowers with larger petals, a consequence of an increase in cell size  <cit> .
this demonstrates that the function of the two crucifer proteins is mainly conserved, whereas that of cyc from the more distantly related species antirrhinum likely diverged  <cit> .
iberis petals are initiated simultaneously as little bulges and the onset of an unequal size development can be detected around the start of stamen differentiation.
from this point on, adaxial and abaxial petals develop differentially throughout flower development.
the major difference in petal size, however, is acquired during late flower development, when a size difference of  <dig> -fold, just after anthesis  increases to  <dig> -fold in fully mature flowers   <cit> .
this raises the question about the molecular network that realises differential petal growth.
comprehensive research has been conducted analysing the genetic basis of general floral organ size determination, which is regulated through several independent pathways .
initial petal growth is achieved through cell proliferation that is later maintained only in restricted regions  <cit> .
growth via cell division ceases and petals acquire their final size through cell elongation, a transition that seems to occur during later stages of flower development, after the maturation of microspores  <cit> .
the switch to cell elongation goes along with an increased expression of cell wall synthesis and cell wall metabolization genes  <cit> .
thus, important determinators of final organ size are factors that control the timing of the cell proliferation arrest and the onset of cell expansion.
the control of plant growth depends, in most cases, on an interplay between different hormones, which can affect either cell division , elongation  or, as auxin does, both processes  <cit> .
growth regulators, like the transcription factors aintegumenta  and jagged, the cytochrome p <dig> monooxygenase kluh or the auxin-inducible gene argos  have a positive effect on petal cell proliferation, mainly via affecting its duration  <cit> .
the restriction of the cell division period during petal development, on the other hand, is accomplished e.g.
through protein degradation by the e <dig> ubiquitin ligase big brother and by the predicted ubiquitin receptor da <dig>  <cit> .
med <dig>  a component of the mediator complex, has a dual function in restricting the periods of cell proliferation as well as cell expansion  <cit> .
similarly, also auxin-response factor <dig> is a negative regulator of cell division early in petal development and represses late cell expansion together with the transcription factor big petal  <cit> .
in contrast, argos like promotes cell expansion  <cit> .
employing rna-seq, a de novo re-assembled petal transcriptome was generated for global analysis of iberis amara petal development and to identify genes differentially expressed in the two petal pairs.
this allowed detailed insight into the molecular network realizing unequal petal growth in the non-model organism iberis amara.
a significantly enhanced occurrence of genes involved in cell wall organization and modification processes in adaxial petals implies that the formation of smaller, adaxial petals is a result of an earlier onset of cell expansion, compared to the large, abaxial petals.
in addition, microarray analyses of transgenic arabidopsis plants ectopically expressing different cyc <dig> transcription factors were performed in order to detect differences in target gene regulation.
RESULTS
analysis of spatial and temporal cell division patterns during iberis amara petal development
both, adaxial and abaxial petals form an upper blade, with roundish conical epidermis cells and a lower claw, with elongated epidermal cells .
elongated claw cells are greenish, indicating the presence of chloroplasts .
after the flowers open, unequal growth continues from stage a <dig>  until the mature flower stage a <dig>  and establishes the major size differences between the two petal pairs, likely via differential cell division, as described by  <cit> .figure  <dig>
iberis amara
petal development.
sem image of adaxial stage a <dig> petal.
adaxial and abaxial stage a <dig>  and stage a <dig>  petals.
: in situ hybridization with iah <dig> of longitudinal sections through iberis amara flower buds before anthesis.
in young flowers, iah <dig> is expressed throughout emerging petal buds.
later, in differentiating petals, iah <dig> localizes to the upper, distal petal part that forms the blade and decreases in the proximal part, developing into the claw.
iah <dig> transcript accumulates before anthesis at petal tips in older flowers.
a few cells scattered throughout the entire petal lengths still express iah <dig>  ad, adaxial; ab, abaxial; b, blade; c, claw; p, petal; se, sepal; lst, lateral stamen; st, stamen; g, gynoecium.
scale bars: a, d-f, 100 μm; b and c, 5 mm.
the temporal and spatial cell division pattern during petal development was analysed by conducting in situ hybridisation studies in younger iberis flower buds before anthesis with the cell cycle marker gene histone <dig> from iberis amara .
initially, iah <dig> is expressed uniformly in petal primordia .
in older flowers, after the start of petal differentiation, iah <dig> expression declines in the proximal region and accumulates in the distal petal part .
this indicates that cell divisions cease first in the proximal region where cells expand into elongated claw cells, while cell divisions in the blade continue.
moreover, there are more cells expressing iah <dig> in the abaxial petal blade compared to adaxial petals , as shown for an earlier stage by  <cit> .
around anthesis, when petals completely cover reproductive structures, iah <dig> expression concentrates predominantly at the distal petal tips and only few iah <dig> expressing cells are detectable in the proximal claw region .
these data show that iberis petals mature along a proximo-distal gradient with a decline in cell division moving from the base to the petal tip.
furthermore, cell division still persists around anthesis in the blade tips, promoting the establishment of the final  <dig> -fold size difference between mature adaxial and abaxial petals.
reconstruction of the iberis amara petal transcriptome
prior to the detection of genes, which are differentially expressed in the iberis amara corolla by rna-seq, a reference petal transcriptome was reconstructed.
two replicates were harvested from adaxial and abaxial petals of stage a <dig> flowers as iatcp <dig> shows at this stage the highest expression in adaxial petals and the largest expression difference between adaxial and abaxial petals  <cit> .
in order to avoid genetic variance due to a lack of isogenic lines from this non-model organism that barely self-pollinates, all petal material was harvested successively from one individual plant.
the illumina sequencing of all four libraries in total produced  <dig> , <dig> quality filtered paired-end reads of approx.
<dig> nucleotides  length, which resembles about  <dig> gb of sequence data.
of those,  <dig> , <dig> reads  were used for the re-assembly , which is comparable to other rna-seq studies  <cit> .
the  <dig>  obtained contigs  could not be extended any more by concatamerization and, thus, represent the petal transcripts used for further analyses.
the average contig size is 677 nt and ranges from  <dig> to  <dig>  nt.table  <dig>
de novo
assembly statistics
a
data
read counts & nucleotides
b
statistics of contigs
c
contig length
number of contigs
%
clean reads from all four libraries, pooled together, were used for the de novo re-assembly of the iberis petal transcriptome.
statistics of re-assembled contigs.
all contigs of the stage a <dig> iberis amara petal transcriptome are classified into categories according to their nucleotide length.
for each category, respective contig numbers and percentage of the total contig number are given.
gb, giga basepairs; nt, nucleotides.
about 59% of all contigs are smaller than  <dig> nucleotides, indicating that likely a larger portion of transcripts is not covered completely by their corresponding contigs.
about 22% of all contigs have a size of up to  <dig>  nt, 14% are between  <dig> - <dig>  nt long, followed by about 5% between  <dig> - <dig>  nt and  <dig> % between  <dig> - <dig>  nt.
only four contigs are larger than  <dig>  nucleotides .
with the established petal transcriptome re-assembly a global analysis of petal development could next be accomplished.
annotation of iberis genes involved in petal development
all  <dig>  contigs were annotated based on blastx searches against an arabidopsis protein database in order to allow a functional categorization of iberis petal transcripts and to facilitate further comparison with arabidopsis expression data.
<dig>  iberis contigs matched to a corresponding arabidopsis genome initiative  code.
<dig> % of matching contigs retrieved an agi code that was found also as a match to other contigs.
this is likely due to the fact that a large proportion of iberis transcripts are not covered completely by their respective contigs and therefore several contigs were assigned to the same agi code.
similarly, detection of an unexpected large number of genes encoding for transcription factors in marchantia by rna-seq was explained as being caused by a “fragmentary” dataset, likely exceeding the number of actual transcription factors present  <cit> .
for this reason, here, contigs with the same agi code were treated as partial transcripts of the same gene and subtracted, yielding  <dig>  putative unigenes.
next, applying an e-value cutoff of e− <dig> resulted in  <dig>   likely unigenes with a putatively conserved function in arabidopsis.
contigs without a hit might be either assembly artefacts or are unique to iberis.
based on their respective agi codes, the  <dig>  iberis unigenes were grouped into functional categories provided by mapman using the classification superviewer tool  <cit> .
for an inter-species transcriptome comparison, microarray data from  <dig>  genes expressed in arabidopsis petals at stage  <dig> was taken.
stage  <dig> starts approximately one day after flower anthesis  <cit>  and is, thus, closest in development to the iberis stage a <dig>  defined as the stage just after anthesis  <cit> .
both transcriptomes show an overall similar distribution of genes grouping into the respective categories, indicating that global crucifer petal development programs are similar .
interestingly, the largest proportion of genes is classified as not assigned.
this class constitutes 28% of the arabidopsis petal transcriptome  and 31% of all transcripts in the monosymmetric corolla of iberis .
in addition, late petal development of both, iberis and arabidopsis, is governed predominantly by genes assigned to the categories protein, rna, signalling, misc , transport and cell, accounting together for about 75% of all categories.figure  <dig>
functional classification of the
iberis amara
and
arabidopsis thaliana
petal transcriptome.
unigenes from iberis and arabidopsis petals were grouped into  <dig> functional categories.
classification of  <dig>  unigenes of the iberis amara stage a <dig> petal transcriptome.
the arabidopsis stage  <dig> petal transcriptome consists of  <dig>  transcripts.
characterization of iberis petal development
in order to analyze gene expression levels in adaxial and abaxial petals, clean reads from the four samples were individually mapped to the de novo re-assembled petal transcriptome.
only reads that uniquely mapped to a single locus of the reference iberis transcriptome were taken into account, amounting to 63–64% of all reads .
gene expression levels are displayed as rpkm  values for each sample library.
after applying a cut-off p-value of  <dig>  for multiplicative errors,  <dig>  genes remained with reliable expression values.
a complete list of all genes with their expression values is given in additional file  <dig> table  <dig>
rna-seq reads and mapping statistics
samples
ad_1
ad_2
ab_1
ab_2
<dig> ,583
<dig> ,334
<dig> ,437
<dig> ,583
all clean reads obtained from the four libraries from adaxial  and abaxial  petals of stage a <dig> iberis flowers were mapped against the re-assembled contigs.
only reads that map uniquely to one locus, highlighted in bold print, were used for the calculation of gene expression.
this applies to 63–64% of total reads from the four libraries.
amongst those  <dig>  transcripts, the top 5% of strongest expressed genes from both petal types were functionally categorized to understand which biological processes are most profoundly required for late petal development in iberis .
the category misc comprises the largest number of highly abundant transcripts.
in addition, petal development seems to be dependent on the strong expression of genes that also exert roles in adaptation to biotic/abiotic stress and in hormone metabolism.
interestingly, transcripts of the aforementioned category not assigned are amongst the top 5% of strongest expressed genes in both adaxial and abaxial iberis petals.
this further corroborates the observation that late petal development seems to require the activity of a large number of thus far uncharacterized genes.table  <dig>
functional categories of most abundant stage a1
iberis
petal transcripts
functional category
number of contigs in
ad petals
ab petals
the 5% highly expressed genes from the  <dig>  iberis amara stage a <dig> adaxial  and abaxial  petals unigenes were assigned to their functional mapman categories.
categories present in both samples are listed, together with the number of genes from each sample that belong to the respective category.
identification of transcripts differentially expressed in adaxial and abaxial petals
for all  <dig>  genes, expression values in adaxial and abaxial petals were compared against each other , resulting in  <dig>  genes being stronger expressed in adaxial petals and  <dig> transcripts with higher rpkm values in abaxial petals.
the majority,  <dig>  genes, have an expression fold change less than 2-fold, with  <dig> being higher expressed in adaxial petals and  <dig> in abaxial petals.
the expression of  <dig> genes is changed two-fold or more between the two petal types, with  <dig> genes being more abundantly transcribed in adaxial petals and only  <dig> genes being more active in abaxial petals.
most of these genes are differentially expressed up to a fold change of five .
only a small fraction displays a fold change equal to or above five , including also iatcp <dig>  which is about 27-times upregulated in adaxial petals, confirming previous observations  <cit>  .table  <dig>
classification and analysis of genes differentially expressed between adaxial and abaxial petals
a
fold change
# genes
↑ adaxial petals
↑ abaxial petals
313
b
functional category
signalling
cell wall
photosynthesis
c
go term
ad petal genes
%
petal genes
%
fold enrichment
all  <dig>  unigenes were classified according to the strength of their expression fold change between adaxial and abaxial petals.
<dig> unigenes, upregulated in adaxial petals , were assigned to their respective functional mapman catagories.
numbers of genes assigned to respective categories are bracketed.
categories that appear also in  are highlighted in bold print.
significantly enriched functional categories amongst  <dig> unigenes, which are upregulated in adaxial petals in comparison to the general iberis petal transcriptome were determined using fatigo  <cit> .
for each go term, the number of genes assigned to the respective go term amongst the  <dig> upregulated adaxial petal genes and the  <dig>  unigenes forming the iberis petal transcriptome, are given.
numbers are also expressed in percent, together with the respective fold enrichment.
overall, the data indicate that a significantly higher gene number governs the development of small adaxial petals compared to the control of large abaxial petal formation.
determination of gene categories in the adaxial petal transcriptome
to get an overview of developmental processes participating in the formation of the two petal types, differentially expressed genes with a minimal fold-change of two were classified into functional mapman categories.
no categories that consisted of more than three members, one of the applied threshold criteria, were found for the  <dig> transcripts upregulated in abaxial petals.
amongst the  <dig> transcripts upregulated in adaxial petals, about 25%  were not assigned into any category, encoding for proteins with unknown functions .
a large number of genes  is grouped together as encoding for various enzymes in the category misc and  <dig> transcripts are involved in protein metabolism, such as protein modification, targeting and degradation.
the category transport comprises  <dig> genes, encoding for proteins transporting inorganic compounds, primary metabolites, ions, metal or auxin.
categories cell wall, signalling and photosynthesis were also detected as being significantly enriched when applying gene ontology  terms of agi codes linked to the adaxial-specific transcripts and are described below.
amongst the category rna ,  <dig> encode for various transcription factors, including iatcp <dig>  in addition,  <dig> adaxial upregulated genes perform a role in the adaptation to biotic and abiotic stress.
to gain a more detailed understanding of the developmental mechanisms and the respective genes involved in generating smaller adaxial petals, significantly enriched go terms amongst the  <dig> adaxial-specific transcripts were revealed using fatigo  <cit> .
individual genes are repeatedly allocated to related go terms.
go terms associated to cell wall-related processes, such as cell wall organization and modification are significantly enriched in adaxial petals .
the in total  <dig> genes exerting cell wall-related functions encode for pectate lyases, pectin methyl esterases/inhibitors implicated in pectin degradation  <cit>  and other enzymes that function in cell wall remodelling and loosening.
even more genes were assigned to the mapman category cell wall   and include, in addition to cell wall degrading/modifying enzymes, cobra like  <dig> and an arabinogalactan protein.
both proteins constitute a putative link between the cell wall and the cytoplasm  <cit> .
the go term cell-cell signalling contains five transcripts encoding for small signal peptides that play a role in plant cell growth .
these peptides were first discovered in tobacco leaf extracts due to their ability to induce a rapid ph increase in cell suspension cultures and were, hence, termed ralfs   <cit> .
the go term flower morphogenesis includes three transcription factors involved in petal development, namely petal loss, blade on petiole  <dig> and  <dig>  the categories endocytosis, membrane budding and clathrin coat assembly include genes that encode for enth/anth/vhs superfamily proteins, key regulators of the endosomal system involved in membrane trafficking  <cit> .
unexpectedly,  <dig> genes were found to be associated with photosynthesis, including the go terms photosynthesis, light harvesting, light reaction and carbon fixation.
in order to analyse whether photosynthesis-related processes contribute to flower monosymmetry formation in iberis, this finding was scrutinized by detailed qpcr analyses.
therefore, a phosphoribulokinase homolog from iberis was analysed as a representative for the category photosynthesis and confirmed the rna-seq data showing a stronger expression in adaxial petals .
next, qpcr was performed on separately harvested petal blade and claw material of the two petal types to test if the greenish claw cells, which are present in both petal types , contribute to the detection of photosynthesis-related genes.
the phosphoribulokinase transcript is not differentially expressed between adaxial and abaxial petal blades or claws , implying that photosynthesis genes do not contribute to the formation of smaller adaxial petals.
in both petal types, the greenish claw region, where the phosphoribulokinase is stronger expressed , is similar in size and shape but blade sizes differ significantly.
therefore, the differential expression of photosynthesis-related genes detected with rna-seq might be due to the fact that harvesting whole petals diluted out claw-expressed genes stronger in large abaxial petals compared to small adaxial petals.
as a consequence, photosynthesis-related genes were excluded from further analyses.
expression analysis of genes upregulated in adaxial iberis petals via qpcr
next, transcripts from other significantly enriched go terms  were further analysed by qpcr.
from the category cell wall organization five representatives with a likely function in cell size control were selected.
all cell wall-related genes were tested exclusively on blade material at stage a <dig> of the two petal types, to exclude effects mediated by elongated claw cells .
all tested iberis genes, a vanguard <dig> homolog  <dig>  a polygalacturonase, a glycosyl hydrolase, a pectate lyase family member and an invertase/pectin methylesterase inhibitor show a  <dig>  to  <dig> -fold stronger expression in adaxial petal blades compared to abaxial ones , implying an enhanced participation of cell wall related processes in the formation of smaller adaxial petals.
from the category cell-cell signalling  two out of the five transcripts were selected for further qpcr analysis.
both iberis ralf genes are stronger expressed in adaxial petals , indicating a possible function of this group of cell-cell signalling genes in the formation of small petal size.
the trihelix transcription factor petal loss  belongs to the significantly enriched category flower morphology  and shows an enhanced expression in adaxial petals .
also, the strong differential petal expression of the tcp transcription factor iatcp <dig>  <cit>  was verified by qpcr and shows the strongest expression difference between the two petal types amongst all genes tested .figure  <dig>
differential expression of cell wall-related genes in adaxial versus abaxial petal blades.
five genes involved in cell wall modification/organization were chosen as representatives for further qpcr analysis:  a vanguard <dig> homolog  <dig>   a polygalacturonase,  a glycosyl hydrolase,  a pectate lyase and  an invertase/pectin methylesterase inhibitor.
stage a <dig> adaxial and abaxial petals from two biological replicates  were dissected into upper blade and lower claw and expression of the respective transcripts was compared only between adaxial and abaxial petal blades.
abpb, abaxial petal blade; adpb, adaxial petal blade.
qpcr of selected genes with a stronger expression in adaxial petals.
expression analysis via qpcr was carried out on adaxial and abaxial petals from two  and three  different biological replicates.
ralf-like  <dig>
ralf-like  <dig>
cob-like  <dig>
petal loss,
iatcp <dig>
p-glycoprotein  <dig>
cytokinin oxidase  <dig>
rcd1-like and  a contig with homology to a protein of unknown function .
abp, abaxial petal; adp, adaxial petal.
from these data, a general picture emerges, where adaxial petals are characterized by a pool of transcripts enriched in genes involved in cell wall organization and signalling.
this implies that smaller petals in comparison to larger petals are the result of an earlier stop of cell division.
concomitantly, up-regulation of genes participating in cell elongation and differentiation occurs earlier in the small, adaxial petals compared to the larger, abaxial petals, the latter remaining longer in the cell proliferation phase.
further genes were selected from transcripts upregulated in adaxial petals  based on their annotation and a putative relation to such processes.
a first interesting candidate for a participation in cell expansion processes is cobra-like  <dig> , a member of the cob multigene family.
cobra, the first analysed member, is strongly expressed in the root elongation zone and was proposed to be a key regulator of oriented cell expansion  <cit> .
similarly, cobl <dig> is involved in the control of the directional and likely female-guided pollen tube growth  <cit> .
in both biological samples, the iberis cobl <dig> is upregulated in adaxial petals compared to abaxial petals , as shown with rna-seq.
another gene upregulated in adaxial iberis petals is p-glycoprotein   <dig> , which belongs to the plant abc transporter superfamily from the category transport .
pgps have been shown to fulfil a role in plant growth and development as they mediate the cellular and long-distance transport of auxin  <cit> .
cytokinin oxidase  <dig>  catalyzes the breakdown of the plant hormone cytokinin and by that exerts a negative influence on cell division  <cit> .
therefore, ckx  <dig> with its rna-seq expression fold change of  <dig>  being slightly below the two-fold threshold was included into further qpcr analysis.
the stronger expression of the iberis ckx  <dig> homolog in adaxial petals  might be involved in reducing the rate of cell division, prior to the switch into cell elongation.
also rcd1-like, a protein involved in cell differentiation from the category rna  shows a higher expression in adaxial petals .
the mammalian rcd <dig> gene is a transcriptional cofactor with a role in the transition of cells to differentiate  <cit> .
the stronger expression in adaxial petals of a gene that shows highest homology to the arabidopsis gene at5g <dig> encoding for a protein of unknown function , confirms the differential expression of a member of this highly abundant class of transcripts upregulated in adaxial petals.
in summary, qpcr analysis strengthens the observation that differential petal size development is accomplished via an earlier onset of cell elongation and differentiation in smaller adaxial petals, as indicated by an upregulation of genes that are involved in cell wall remodelling, repression of cell division, cell differentiation and signalling.
interspecies comparison of cyc <dig> target genes via ectopic cyc <dig> expression in the heterologous system arabidopsis thaliana
transgenic arabidopsis plants overexpressing the two crucifer cyc <dig> transcription factors iatcp <dig> and tcp <dig> produce flowers with smaller petals, whereas ectopic expression of cyc from antirrhinum results in the formation of larger petals  <cit> .
to compare the effect of the cyc, iatcp <dig> and tcp <dig> proteins on downstream regulatory networks in arabidopsis, a microarray analysis was conducted.
material for rna extraction was harvested from young inflorescences, comprising flower bud stages before anthesis, of transgenic t <dig> plants overexpressing these genes.
in total,  <dig> genes were determined to be differentially regulated following ectopic iatcp <dig> expression, the majority  being negatively affected .
slightly less genes  respond to an overexpression of cyc .
the lowest number of genes  is addressed by ectopic tcp <dig> activity .figure  <dig>
numbers of differentially expressed genes caused by ectopic activity of
cyc2
transcription factors.
the diagram shows the numbers of differentially expressed genes detected with microarrays hybridized with inflorescence cdna from transgenic arabidopsis plants overexpressing either iatcp <dig> , cyc  or tcp <dig> , together with respective floral phenotypes.
total target gene numbers are given outside the circles, numbers of target genes solely addressed by respective transcription factors are shown inside the circles.
numbers of genes that are mutually addressed by two or all three cyc <dig> transcription factors are displayed in intersections.
arrows indicate up- or downregulation, asterisks denote mutually addressed genes with a reciprocal expression.
scale bars: 2 mm.
the cyc <dig> transcription factors cyc and iatcp <dig>  which govern monosymmetry development, address both a larger number of target genes in the heterologous arabidopsis system than tcp <dig>  this tendency is more obvious regarding only the number of target genes, which are exclusively addressed by a single cyc <dig> protein, namely  <dig> by cyc,  <dig> by iatcp <dig> and only  <dig> by tcp <dig>  the capability of the cyc and iatcp <dig> genes to address a larger number of downstream targets might reflect their function in the control of monosymmetry formation, even when expressed in a heterologous species forming a polysymmetric corolla such as arabidopsis.
<dig> target genes are commonly addressed by all three transcription factors and might thus not be linked to specific functions in symmetry regulation.
the highest number of overlappingly regulated genes is found between the two crucifer transcription factors , whereas the smallest number of targets is mutually addressed by cyc and tcp <dig> .
to gain insight into the molecular network acting downstream of iatcp <dig>  significantly accumulated go terms were detected.
for the  <dig> genes repressed by iatcp <dig>  the majority of enriched go terms are associated with cell wall-related processes .
other enriched go terms are associated with secondary metabolic processes, h2o2catabolic processes and response to red light .
additional file  <dig> summarizes single genes, amongst them  <dig> cell wall genes that are mutually addressed by ectopic expression of iatcp <dig> in arabidopsis inflorescences and by endogenous iatcp <dig> in adaxial iberis petals.table  <dig>
enriched go terms in the pool of
iatcp1
target genes in
arabidopsis
inflorescences
go term
genes ↓ by iatcp1
%
arabidopsis infl.
genes
%
fold enrichment
listed are go terms that are significantly enriched amongst all genes negatively regulated upon iatcp <dig> overexpression in arabidopsis inflorescences in comparison to the arabidopsis inflorescence transcriptome.
for each go term, numbers of genes clustering to these categories from the pools of  <dig> downregulated genes and from the  <dig>  genes forming the arabidopsis inflorescence transcriptome, are given.
numbers are also expressed in percent, together with their respective fold enrichment.
rna-seq and microarray analyses, both, detected a differential regulation of genes involved in cell wall processes.
however, in young transgenic arabidopsis inflorescences all cell wall-related transcripts are downregulated following an ectopic iatcp <dig> activity, whereas they are upregulated in older iberis petals after anthesis, in correlation with and presumably also in response to a high endogenous iatcp1-expression.
this indicates that the spatial and temporal context of cyc <dig> activity is crucial for its proper effect on cell growth and differentiation.
discussion
monosymmetry in iberis is achieved through differential cell proliferation during late flower development
iberis petals are initiated simultaneously and unequal growth begins around the onset of stamen differentiation, from where it proceeds and gains its maximal size difference after anthesis, likely driven by differential cell proliferation  <cit> .
this requires cell division to continue throughout later phases of flower development, which was demonstrated by monitoring the cell division pattern via histone <dig> expression from iberis in different petal stages.
in older petals, cell division still takes place close to anthesis in both petal types, where it is restricted mainly to the petal tips.
similar to iberis, cell division in arabidopsis petals increases throughout flower development until stage  <dig> and can still be visualized in stage  <dig>  just before anthesis  <cit> .
after anthesis, adaxial iberis petals increase  <dig> -fold, whereas abaxial petals increase  <dig> -fold to reach their final size.
in contrast, cells of both petal types only double in size from anthesis to maturity  <cit> .
these data indicate that cell division – at least in abaxial petals – proceeds beyond anthesis.
transcriptome analysis of petal development of iberis amara
rna-seq is being used more and more often as a convenient tool for comparative transcriptome analyses of species with inavailable genome sequences  <cit> .
applying an rna-seq approach, we investigated the iberis transcriptome at the late petal developmental stage a <dig>  just after anthesis of flowers.
comparison of the arabidopsis and iberis petal transcript distribution into functional categories indicates that molecular processes governing late petal development are similar in the two crucifers.
interestingly, for the largest group of the iberis petal transcriptome, comprising 31% of the transcripts, no function could be assigned yet.
a similar situation with 28% of these genes is observed for arabidopsis.
interestingly, comparative analysis of different petal stages in antirrhinum, where flowers form an even more elaborate and complex shaped corolla, revealed that the frequency of the unclassified protein group accounts for over 50% amongst all transcripts expressed in petals  <cit> .
this suggests that a large proportion of genes exert an intriguing, yet uncharacterized, function during petal morphogenesis.
analysis of the most abundant transcripts in iberis petal stage a <dig> revealed that various enzymes, subsumed in the category misc, are highly expressed in the two petal types, amongst them gdsl-lipases and lipid transfer proteins.
both enzyme types have a function in lipid metabolism and have been described as putative target genes of the petal and stamen-specifying b-class mads box transcription factor during petal development in antirrhinum and arabidopsis  <cit> .
the cytochrome p <dig> gene cyp76c <dig>  which is amongst the most highly expressed iberis petal genes, is also strongly expressed in various arabidopsis floral organs, where it participates in the linalool metabolism.
linalool, a well-studied attractant for pollinators, is converted further into its oxigenated derivates by cyp76c <dig> in autogamous arabidopsis flowers  <cit> , which likely function in plant defence against pathogens and insect herbivores  <cit> .
two lipoxygenases, lox <dig> and lox <dig>  which exert functions in hormone metabolism, are also highly expressed in iberis petals.
the lox <dig> protein catalyzes the first dedicated step in jasmonic acid  biosynthesis and is activated by the transcription factor tcp <dig> in leaves  <cit> .
tcp <dig> is also expressed in both iberis petal types , and since lox <dig> is localized to chloroplasts  <cit> , lox2-dependent induction of ja likely takes place in the green petal claws, where ja might exert a negative effect on cell proliferation  <cit> .
the monosymmetric corolla formation of iberis is realized through differential timing of the switch from cell division to cell elongation
with almost 15-times more genes upregulated in the small adaxial petals compared to the large abaxial petals, it seems that more genes are required to restrict adaxial petal size during iberis corolla development.
the fact that the monosymmetry key regulator iatcp <dig> is highly expressed in adaxial petals and represents one of the genes with the largest expression difference implies that iatcp <dig> activates a larger number of target genes compared to its inhibitory transcription effects.
differential iberis petal development is likely driven by unequal cell division processes.
consistently, genes exerting negative effects on cell division are amongst the pool of transcripts upregulated in adaxial petals.
in arabidopsis, cytokinin oxidase   <dig> catalyses the metabolic breakdown of the plant hormone cytokinin and plants with defective ckx genes form flowers with enlarged petals due to an increased cell number  <cit> .
the trihelix transcription factor ptl is expressed in regions of developing arabidopsis flowers, where growth is repressed.
ptl expression in the inter-sepal zone is assumed to keep cell proliferation under control to allow the outgrowth of petal primordia.
the loss of ptl function results in an increased cell proliferation in that region  <cit> .
petals are initiated in response to localized auxin accumulation, and lampugnani and colleagues  <cit>  speculate this could be evoked via a supportive role of ptl in non-polar auxin efflux through pgps.
pgps belong to the superfamily of abc-transporters and function in mediating auxin transport  <cit> .
pgp <dig>  which is also transcriptionally enriched in adaxial iberis petals, belongs to the same clade as the auxin exporters pgp <dig> and pgp <dig>  <cit> , for which double mutant plants show severe growth defects  <cit> .
in addition to transcripts negatively affecting cell proliferation, a large number of genes related to cell wall modifying processes are upregulated in adaxial petals.
after an initial phase of organ growth by cell division, final organ size is realised by cell elongation  <cit> .
the primary cell wall of land plants is composed of a strong cellulose microfibril framework cross-linked through xyloglucan  chains and embedded in a stabilizing matrix of pectin polysaccharides and structural proteins.
plant cell walls provide stability by withstanding tensile stress, and cell growth results from changes in wall extensibility by relenting to this stress  <cit> .
amongst the group of cell wall modifying proteins in adaxial iberis petals are several pectinases, enzymes that function in the de-/methylesterification or degradation of pectin  <cit>  and, by that, might contribute to cell growth via precisely tuning the proportion of pectate in the cell wall  <cit> .
a xyloglucan endotransglucosylase , which cleaves and re-attaches xyloglucan chains in vitro was found to be upregulated in adaxial petals.
xet activity correlates with regions undergoing cell expansion, indicating that xets have a role in cell expansion, e.g.
through the biogenesis of newly formed cell walls .
in addition, several iberis glycosyl hydrolases  were upregulated in adaxial petals, which form a diverse group of enzymes that hydrolyze glycosidic bonds  <cit> .
one arabidopsis homolog of the ghs belongs to the subfamily of the endo- <dig> -ß-glucanases korrigan, which is essential for cell elongation and cellulose synthesis in arabidopsis hypocotyls  <cit> .
several transcripts encoding for a cobra-like protein, two proline rich extension-like receptor kinases , an arabinogalactan protein and a formin are upregulated in adaxial petals and might act as a potential connection between the cytoplasma and cell wall.
these proteins are, thus, part of the molecular continuum that connects the cytoskeletal network, the cytoplasm and the cell wall, and which contributes to cell expansion control in arabidopsis .
arabidopsis homologs of several of the floral cell wall-related genes, such as a polygalacturonase , a xet  and a pectin lyase like member  are also predominately expressed in the cell elongation zone of root tips  and only weakly in the preceeding meristematic region, where growth is realized by cell divisions.
this supports their general function in cell wall modification, both in a floral and root context, contributing to tissue growth by mediating cell elongation processes.
the determination of final organ size requires the precise coordination of both, cell division and cell elongation processes, and likely depends on a tight cellular communication.
amongst all adaxial-petal specific genes, a group of transcripts involved in cell-cell signalling is significantly enriched.
these ralfs are small peptides, which are important for many developmental processes and regarded as novel plant hormones  <cit> .
in grass leaves, ralfs were found to be mainly expressed in young elongating cells beyond the meristematic zone, and expression is almost absent in mature cells  <cit> .
as ralfs are thought to exert a negative effect on cell growth, their expression strength and location is likely precisely adjusted during organ development to achieve an accurate guidance of cell expansion without completely blocking it  <cit> .
summing up, a significantly increased number of transcripts involved in cell wall modification and cell-cell signalling, together with genes regulating the restriction of cell division and auxin transport were detected in the pool of  <dig> transcripts enriched in small, adaxial iberis petals.
hence, unequal petal growth is likely achieved via an earlier stop of cell division in adaxial petals with a subsequent earlier onset of cell expansion, whilst abaxial petal cells continue dividing.
in iberis, cell division ceases in an acropetal gradient, where cells at the tip keep on dividing longest, similar as in other plant species  <cit> .
thus, an unequal stop of cell division might be realised through a faster acropetal progression of a cell division stop front in adaxial petals compared to abaxial petals.
ectopic expression of iatcp <dig> causes expression changes of cell wall related genes in arabidopsis
microarray analyses aimed at comparing the molecular networks that are addressed in inflorescences by iatcp <dig> and tcp <dig> causing smaller petal formation, and by cyc, giving rise to the development of larger petals.
a considerable number of genes  is addressed by all three cyc <dig> proteins together.
these might be transcripts that constitute a common basal set of tcp/cyc <dig> transcription factor targets genes in arabidopsis thaliana.
interestingly, in the heterologous arabidopsis system, more genes are exclusively addressed by iatcp <dig> from iberis  and cyc from antirrhinum , developing a monosymmetric flower, than by tcp <dig> from arabidopsis , forming a polysymmetric corolla.
these quantitative differences indicate that iatcp <dig> and cyc might regulate more target genes, implying that a larger downstream regulatory network realizes monosymmetric compared to polysymmetric corolla development.
therefore, in addition to cis-regulatory changes that led to a heterochronic cyc <dig> gene expression shift in the brassicaceae, promoting corolla monosymmetry evolution  <cit> , also a divergence of trans-acting cyc <dig> proteins likely contributed to the establishment of this morphological novelty.
the largest overlap in target gene number is shared between iatcp <dig> and tcp <dig>  these  <dig> genes comprise several transcripts involved in cell wall-related and signalling processes  and the activity of these exclusive crucifer targets could be essential for the petal size restriction in arabidopsis, as only transgenic plants ectopically expressing a crucifer cyc <dig> protein develop flowers with smaller petals  <cit> .
amongst the population of target genes downregulated in response to heterologous iatcp <dig> expression in arabidopsis, again a high proportion is involved in cell wall modification processes.
this indicates that iatcp <dig> addresses these genes in arabidopsis and that the high abundance of this gene class detected with rna-seq in adaxial iberis petals is the effect of the strong iatcp <dig> expression there.
while cell wall genes are positively affected in older adaxial iberis petals, where iatcp <dig> expression is strongly enhanced, iatcp <dig> activity has a negative effect on cell wall genes in young arabidopsis inflorescences overexpressing iatcp <dig>  tcp transcription factors are known to exert either an activating or repressive effect on organ growth, depending on their tissue specific expression  <cit> .
as tcp proteins act as dimers  <cit> , the outcome of their activity might differ in dependence of available interaction partners in different organs or developmental stages.
similar development-dependent regulatory effects were shown previously for the knox/bel transcription factor bellringer  <cit>  , which interacts with other bel and knox proteins  <cit> .
one of its targets, the pectin methylesterase pme <dig> is repressed by blr in the shoot meristem, to allow coordinated formation of primordia, but is activated by blr in internodes, where it promotes cell elongation  <cit> .
CONCLUSIONS
an rna-seq expression analysis on adaxial and abaxial stage a <dig> iberis petals reveals a significantly increased number of cell wall-related transcripts in adaxial petals.
this indicates that differential petal growth, a characteristic of the monosymmetric iberis corolla, is achieved through an earlier stop of the cell proliferation phase followed by the onset of cell expansion and differentiation in the smaller adaxial petals, while abaxial petal cells likely still continue growth via cell division.
as ectopic expression of iatcp <dig> in arabidopsis addresses cell wall-related genes, the enhanced occurrence of this gene class in adaxial iberis petals is very likely also a direct consequence of the strong iatcp <dig> expression there.
moreover, crucifer monosymmetry evolution likely relied on the combination of cis-regulatory changes, leading to a late adaxial iatcp <dig> expression and also on trans-regulatory changes mediated by differences in the cyc <dig> proteins themselves, altering the set of potential target genes that contribute to the large petal size differences in iberis amara.

 in the co-evolution between plants and insects, the development of floral monosymmetry was an important step, facilitating angiosperm speciation as a response to the adaptation to the visual senses of insect pollinators. monosymmetry, thus, likely functioned as a morphological key innovation and evolved independently in different angiosperm lineages. groundbreaking research almost two decades ago identified the tcp transcription factor cycloidea as the molecular key regulator of monosymmetry development in antirrhinum. cyc and its paralog dichotoma are expressed in the adaxial part of developing antirrhinum flowers, where they guide the acquisition of adaxial identities of second and third whorl organs. cyc belongs to the cyc clade of the tcp transcription factor family and in all core eudicot species analysed so far, monosymmetry development is controlled by cyc clade genes. the majority of crucifers develop a polysymmetric corolla and only six genera form flowers with two petal pairs of different sizes. in iberis amara, unequal petal pair formation correlates with a stronger expression of the cyc clade gene iatcp in the smaller, adaxial petals. comparison of adaxial and abaxial epidermal cell sizes revealed that petal size differences are due to a differential rate of cell proliferation. in a peloric flower variant, forming only large, abaxialized petals, the iatcp expression is dramatically decreased. transgenic arabidopsis plants overexpressing the cruciferous cyc transcription factors iatcp from iberis amara and tcp from arabidopsis, both, produce similar flowers with smaller petals. for plants overexpressing iatcp this was shown to be due to a reduction in cell number. contrarily, ectopic expression of cyc from antirrhinum results in transgenic plants forming flowers with larger petals, a consequence of an increase in cell size. this demonstrates that the function of the two crucifer proteins is mainly conserved, whereas that of cyc from the more distantly related species antirrhinum likely diverged. iberis petals are initiated simultaneously as little bulges and the onset of an unequal size development can be detected around the start of stamen differentiation. from this point on, adaxial and abaxial petals develop differentially throughout flower development. the major difference in petal size, however, is acquired during late flower development, when a size difference of -fold, just after anthesis increases to -fold in fully mature flowers. this raises the question about the molecular network that realises differential petal growth. comprehensive research has been conducted analysing the genetic basis of general floral organ size determination, which is regulated through several independent pathways. initial petal growth is achieved through cell proliferation that is later maintained only in restricted regions. growth via cell division ceases and petals acquire their final size through cell elongation, a transition that seems to occur during later stages of flower development, after the maturation of microspores. the switch to cell elongation goes along with an increased expression of cell wall synthesis and cell wall metabolization genes. thus, important determinators of final organ size are factors that control the timing of the cell proliferation arrest and the onset of cell expansion. the control of plant growth depends, in most cases, on an interplay between different hormones, which can affect either cell division, elongation or, as auxin does, both processes. growth regulators, like the transcription factors aintegumenta and jagged, the cytochrome p monooxygenase kluh or the auxin-inducible gene argos have a positive effect on petal cell proliferation, mainly via affecting its duration. the restriction of the cell division period during petal development, on the other hand, is accomplished e.g. through protein degradation by the e ubiquitin ligase big brother and by the predicted ubiquitin receptor da. med a component of the mediator complex, has a dual function in restricting the periods of cell proliferation as well as cell expansion. similarly, also auxin-response factor is a negative regulator of cell division early in petal development and represses late cell expansion together with the transcription factor big petal. in contrast, argos like promotes cell expansion. employing rna-seq, a de novo re-assembled petal transcriptome was generated for global analysis of iberis amara petal development and to identify genes differentially expressed in the two petal pairs. this allowed detailed insight into the molecular network realizing unequal petal growth in the non-model organism iberis amara. a significantly enhanced occurrence of genes involved in cell wall organization and modification processes in adaxial petals implies that the formation of smaller, adaxial petals is a result of an earlier onset of cell expansion, compared to the large, abaxial petals. in addition, microarray analyses of transgenic arabidopsis plants ectopically expressing different cyc transcription factors were performed in order to detect differences in target gene regulation.  analysis of spatial and temporal cell division patterns during iberis amara petal development both, adaxial and abaxial petals form an upper blade, with roundish conical epidermis cells and a lower claw, with elongated epidermal cells. elongated claw cells are greenish, indicating the presence of chloroplasts. after the flowers open, unequal growth continues from stage a until the mature flower stage a and establishes the major size differences between the two petal pairs, likely via differential cell division, as described by.figure iberis amara petal development. sem image of adaxial stage a petal. adaxial and abaxial stage a and stage a petals.: in situ hybridization with iah of longitudinal sections through iberis amara flower buds before anthesis. in young flowers, iah is expressed throughout emerging petal buds. later, in differentiating petals, iah localizes to the upper, distal petal part that forms the blade and decreases in the proximal part, developing into the claw. iah transcript accumulates before anthesis at petal tips in older flowers. a few cells scattered throughout the entire petal lengths still express iah ad, adaxial; ab, abaxial; b, blade; c, claw; p, petal; se, sepal; lst, lateral stamen; st, stamen; g, gynoecium. scale bars: a, d-f, 100 μm; b and c, 5 mm. the temporal and spatial cell division pattern during petal development was analysed by conducting in situ hybridisation studies in younger iberis flower buds before anthesis with the cell cycle marker gene histone from iberis amara. initially, iah is expressed uniformly in petal primordia. in older flowers, after the start of petal differentiation, iah expression declines in the proximal region and accumulates in the distal petal part. this indicates that cell divisions cease first in the proximal region where cells expand into elongated claw cells, while cell divisions in the blade continue. moreover, there are more cells expressing iah in the abaxial petal blade compared to adaxial petals, as shown for an earlier stage by. around anthesis, when petals completely cover reproductive structures, iah expression concentrates predominantly at the distal petal tips and only few iah expressing cells are detectable in the proximal claw region. these data show that iberis petals mature along a proximo-distal gradient with a decline in cell division moving from the base to the petal tip. furthermore, cell division still persists around anthesis in the blade tips, promoting the establishment of the final -fold size difference between mature adaxial and abaxial petals. reconstruction of the iberis amara petal transcriptome prior to the detection of genes, which are differentially expressed in the iberis amara corolla by rna-seq, a reference petal transcriptome was reconstructed. two replicates were harvested from adaxial and abaxial petals of stage a flowers as iatcp shows at this stage the highest expression in adaxial petals and the largest expression difference between adaxial and abaxial petals. in order to avoid genetic variance due to a lack of isogenic lines from this non-model organism that barely self-pollinates, all petal material was harvested successively from one individual plant. the illumina sequencing of all four libraries in total produced, quality filtered paired-end reads of approx. nucleotides length, which resembles about gb of sequence data. of those,, reads were used for the re-assembly, which is comparable to other rna-seq studies. the obtained contigs could not be extended any more by concatamerization and, thus, represent the petal transcripts used for further analyses. the average contig size is 677 nt and ranges from to  nt.table de novo assembly statistics a data read counts & nucleotides b statistics of contigs c contig length number of contigs % clean reads from all four libraries, pooled together, were used for the de novo re-assembly of the iberis petal transcriptome. statistics of re-assembled contigs. all contigs of the stage a iberis amara petal transcriptome are classified into categories according to their nucleotide length. for each category, respective contig numbers and percentage of the total contig number are given. gb, giga basepairs; nt, nucleotides. about 59% of all contigs are smaller than nucleotides, indicating that likely a larger portion of transcripts is not covered completely by their corresponding contigs. about 22% of all contigs have a size of up to  nt, 14% are between -  nt long, followed by about 5% between -  nt and % between -  nt. only four contigs are larger than nucleotides. with the established petal transcriptome re-assembly a global analysis of petal development could next be accomplished. annotation of iberis genes involved in petal development all contigs were annotated based on blastx searches against an arabidopsis protein database in order to allow a functional categorization of iberis petal transcripts and to facilitate further comparison with arabidopsis expression data. iberis contigs matched to a corresponding arabidopsis genome initiative code. % of matching contigs retrieved an agi code that was found also as a match to other contigs. this is likely due to the fact that a large proportion of iberis transcripts are not covered completely by their respective contigs and therefore several contigs were assigned to the same agi code. similarly, detection of an unexpected large number of genes encoding for transcription factors in marchantia by rna-seq was explained as being caused by a “fragmentary” dataset, likely exceeding the number of actual transcription factors present. for this reason, here, contigs with the same agi code were treated as partial transcripts of the same gene and subtracted, yielding putative unigenes. next, applying an e-value cutoff of e− resulted in likely unigenes with a putatively conserved function in arabidopsis. contigs without a hit might be either assembly artefacts or are unique to iberis. based on their respective agi codes, the iberis unigenes were grouped into functional categories provided by mapman using the classification superviewer tool. for an inter-species transcriptome comparison, microarray data from genes expressed in arabidopsis petals at stage was taken. stage starts approximately one day after flower anthesis and is, thus, closest in development to the iberis stage a defined as the stage just after anthesis. both transcriptomes show an overall similar distribution of genes grouping into the respective categories, indicating that global crucifer petal development programs are similar. interestingly, the largest proportion of genes is classified as not assigned. this class constitutes 28% of the arabidopsis petal transcriptome and 31% of all transcripts in the monosymmetric corolla of iberis. in addition, late petal development of both, iberis and arabidopsis, is governed predominantly by genes assigned to the categories protein, rna, signalling, misc, transport and cell, accounting together for about 75% of all categories.figure functional classification of the iberis amara and arabidopsis thaliana petal transcriptome. unigenes from iberis and arabidopsis petals were grouped into functional categories. classification of unigenes of the iberis amara stage a petal transcriptome. the arabidopsis stage petal transcriptome consists of transcripts. characterization of iberis petal development in order to analyze gene expression levels in adaxial and abaxial petals, clean reads from the four samples were individually mapped to the de novo re-assembled petal transcriptome. only reads that uniquely mapped to a single locus of the reference iberis transcriptome were taken into account, amounting to 63–64% of all reads. gene expression levels are displayed as rpkm values for each sample library. after applying a cut-off p-value of for multiplicative errors, genes remained with reliable expression values. a complete list of all genes with their expression values is given in additional file table rna-seq reads and mapping statistics samples ad_1 ad_2 ab_1 ab_2,583,334,437,583 all clean reads obtained from the four libraries from adaxial and abaxial petals of stage a iberis flowers were mapped against the re-assembled contigs. only reads that map uniquely to one locus, highlighted in bold print, were used for the calculation of gene expression. this applies to 63–64% of total reads from the four libraries. amongst those transcripts, the top 5% of strongest expressed genes from both petal types were functionally categorized to understand which biological processes are most profoundly required for late petal development in iberis. the category misc comprises the largest number of highly abundant transcripts. in addition, petal development seems to be dependent on the strong expression of genes that also exert roles in adaptation to biotic/abiotic stress and in hormone metabolism. interestingly, transcripts of the aforementioned category not assigned are amongst the top 5% of strongest expressed genes in both adaxial and abaxial iberis petals. this further corroborates the observation that late petal development seems to require the activity of a large number of thus far uncharacterized genes.table functional categories of most abundant stage a1 iberis petal transcripts functional category number of contigs in ad petals ab petals the 5% highly expressed genes from the iberis amara stage a adaxial and abaxial petals unigenes were assigned to their functional mapman categories. categories present in both samples are listed, together with the number of genes from each sample that belong to the respective category. identification of transcripts differentially expressed in adaxial and abaxial petals for all genes, expression values in adaxial and abaxial petals were compared against each other, resulting in genes being stronger expressed in adaxial petals and transcripts with higher rpkm values in abaxial petals. the majority, genes, have an expression fold change less than 2-fold, with being higher expressed in adaxial petals and in abaxial petals. the expression of genes is changed two-fold or more between the two petal types, with genes being more abundantly transcribed in adaxial petals and only genes being more active in abaxial petals. most of these genes are differentially expressed up to a fold change of five. only a small fraction displays a fold change equal to or above five, including also iatcp which is about 27-times upregulated in adaxial petals, confirming previous observations.table classification and analysis of genes differentially expressed between adaxial and abaxial petals a fold change # genes ↑ adaxial petals ↑ abaxial petals 313 b functional category signalling cell wall photosynthesis c go term ad petal genes % petal genes % fold enrichment all unigenes were classified according to the strength of their expression fold change between adaxial and abaxial petals. unigenes, upregulated in adaxial petals, were assigned to their respective functional mapman catagories. numbers of genes assigned to respective categories are bracketed. categories that appear also in are highlighted in bold print. significantly enriched functional categories amongst unigenes, which are upregulated in adaxial petals in comparison to the general iberis petal transcriptome were determined using fatigo. for each go term, the number of genes assigned to the respective go term amongst the upregulated adaxial petal genes and the unigenes forming the iberis petal transcriptome, are given. numbers are also expressed in percent, together with the respective fold enrichment. overall, the data indicate that a significantly higher gene number governs the development of small adaxial petals compared to the control of large abaxial petal formation. determination of gene categories in the adaxial petal transcriptome to get an overview of developmental processes participating in the formation of the two petal types, differentially expressed genes with a minimal fold-change of two were classified into functional mapman categories. no categories that consisted of more than three members, one of the applied threshold criteria, were found for the transcripts upregulated in abaxial petals. amongst the transcripts upregulated in adaxial petals, about 25% were not assigned into any category, encoding for proteins with unknown functions. a large number of genes is grouped together as encoding for various enzymes in the category misc and transcripts are involved in protein metabolism, such as protein modification, targeting and degradation. the category transport comprises genes, encoding for proteins transporting inorganic compounds, primary metabolites, ions, metal or auxin. categories cell wall, signalling and photosynthesis were also detected as being significantly enriched when applying gene ontology terms of agi codes linked to the adaxial-specific transcripts and are described below. amongst the category rna, encode for various transcription factors, including iatcp in addition, adaxial upregulated genes perform a role in the adaptation to biotic and abiotic stress. to gain a more detailed understanding of the developmental mechanisms and the respective genes involved in generating smaller adaxial petals, significantly enriched go terms amongst the adaxial-specific transcripts were revealed using fatigo. individual genes are repeatedly allocated to related go terms. go terms associated to cell wall-related processes, such as cell wall organization and modification are significantly enriched in adaxial petals. the in total genes exerting cell wall-related functions encode for pectate lyases, pectin methyl esterases/inhibitors implicated in pectin degradation and other enzymes that function in cell wall remodelling and loosening. even more genes were assigned to the mapman category cell wall and include, in addition to cell wall degrading/modifying enzymes, cobra like and an arabinogalactan protein. both proteins constitute a putative link between the cell wall and the cytoplasm. the go term cell-cell signalling contains five transcripts encoding for small signal peptides that play a role in plant cell growth. these peptides were first discovered in tobacco leaf extracts due to their ability to induce a rapid ph increase in cell suspension cultures and were, hence, termed ralfs. the go term flower morphogenesis includes three transcription factors involved in petal development, namely petal loss, blade on petiole and the categories endocytosis, membrane budding and clathrin coat assembly include genes that encode for enth/anth/vhs superfamily proteins, key regulators of the endosomal system involved in membrane trafficking. unexpectedly, genes were found to be associated with photosynthesis, including the go terms photosynthesis, light harvesting, light reaction and carbon fixation. in order to analyse whether photosynthesis-related processes contribute to flower monosymmetry formation in iberis, this finding was scrutinized by detailed qpcr analyses. therefore, a phosphoribulokinase homolog from iberis was analysed as a representative for the category photosynthesis and confirmed the rna-seq data showing a stronger expression in adaxial petals. next, qpcr was performed on separately harvested petal blade and claw material of the two petal types to test if the greenish claw cells, which are present in both petal types, contribute to the detection of photosynthesis-related genes. the phosphoribulokinase transcript is not differentially expressed between adaxial and abaxial petal blades or claws, implying that photosynthesis genes do not contribute to the formation of smaller adaxial petals. in both petal types, the greenish claw region, where the phosphoribulokinase is stronger expressed, is similar in size and shape but blade sizes differ significantly. therefore, the differential expression of photosynthesis-related genes detected with rna-seq might be due to the fact that harvesting whole petals diluted out claw-expressed genes stronger in large abaxial petals compared to small adaxial petals. as a consequence, photosynthesis-related genes were excluded from further analyses. expression analysis of genes upregulated in adaxial iberis petals via qpcr next, transcripts from other significantly enriched go terms were further analysed by qpcr. from the category cell wall organization five representatives with a likely function in cell size control were selected. all cell wall-related genes were tested exclusively on blade material at stage a of the two petal types, to exclude effects mediated by elongated claw cells. all tested iberis genes, a vanguard homolog a polygalacturonase, a glycosyl hydrolase, a pectate lyase family member and an invertase/pectin methylesterase inhibitor show a to -fold stronger expression in adaxial petal blades compared to abaxial ones, implying an enhanced participation of cell wall related processes in the formation of smaller adaxial petals. from the category cell-cell signalling two out of the five transcripts were selected for further qpcr analysis. both iberis ralf genes are stronger expressed in adaxial petals, indicating a possible function of this group of cell-cell signalling genes in the formation of small petal size. the trihelix transcription factor petal loss belongs to the significantly enriched category flower morphology and shows an enhanced expression in adaxial petals. also, the strong differential petal expression of the tcp transcription factor iatcp was verified by qpcr and shows the strongest expression difference between the two petal types amongst all genes tested.figure differential expression of cell wall-related genes in adaxial versus abaxial petal blades. five genes involved in cell wall modification/organization were chosen as representatives for further qpcr analysis: a vanguard homolog a polygalacturonase, a glycosyl hydrolase, a pectate lyase and an invertase/pectin methylesterase inhibitor. stage a adaxial and abaxial petals from two biological replicates were dissected into upper blade and lower claw and expression of the respective transcripts was compared only between adaxial and abaxial petal blades. abpb, abaxial petal blade; adpb, adaxial petal blade. qpcr of selected genes with a stronger expression in adaxial petals. expression analysis via qpcr was carried out on adaxial and abaxial petals from two and three different biological replicates. ralf-like ralf-like cob-like petal loss, iatcp p-glycoprotein cytokinin oxidase rcd1-like and a contig with homology to a protein of unknown function. abp, abaxial petal; adp, adaxial petal. from these data, a general picture emerges, where adaxial petals are characterized by a pool of transcripts enriched in genes involved in cell wall organization and signalling. this implies that smaller petals in comparison to larger petals are the result of an earlier stop of cell division. concomitantly, up-regulation of genes participating in cell elongation and differentiation occurs earlier in the small, adaxial petals compared to the larger, abaxial petals, the latter remaining longer in the cell proliferation phase. further genes were selected from transcripts upregulated in adaxial petals based on their annotation and a putative relation to such processes. a first interesting candidate for a participation in cell expansion processes is cobra-like, a member of the cob multigene family. cobra, the first analysed member, is strongly expressed in the root elongation zone and was proposed to be a key regulator of oriented cell expansion. similarly, cobl is involved in the control of the directional and likely female-guided pollen tube growth. in both biological samples, the iberis cobl is upregulated in adaxial petals compared to abaxial petals, as shown with rna-seq. another gene upregulated in adaxial iberis petals is p-glycoprotein, which belongs to the plant abc transporter superfamily from the category transport. pgps have been shown to fulfil a role in plant growth and development as they mediate the cellular and long-distance transport of auxin. cytokinin oxidase catalyzes the breakdown of the plant hormone cytokinin and by that exerts a negative influence on cell division. therefore, ckx with its rna-seq expression fold change of being slightly below the two-fold threshold was included into further qpcr analysis. the stronger expression of the iberis ckx homolog in adaxial petals might be involved in reducing the rate of cell division, prior to the switch into cell elongation. also rcd1-like, a protein involved in cell differentiation from the category rna shows a higher expression in adaxial petals. the mammalian rcd gene is a transcriptional cofactor with a role in the transition of cells to differentiate. the stronger expression in adaxial petals of a gene that shows highest homology to the arabidopsis gene at5g encoding for a protein of unknown function, confirms the differential expression of a member of this highly abundant class of transcripts upregulated in adaxial petals. in summary, qpcr analysis strengthens the observation that differential petal size development is accomplished via an earlier onset of cell elongation and differentiation in smaller adaxial petals, as indicated by an upregulation of genes that are involved in cell wall remodelling, repression of cell division, cell differentiation and signalling. interspecies comparison of cyc target genes via ectopic cyc expression in the heterologous system arabidopsis thaliana transgenic arabidopsis plants overexpressing the two crucifer cyc transcription factors iatcp and tcp produce flowers with smaller petals, whereas ectopic expression of cyc from antirrhinum results in the formation of larger petals. to compare the effect of the cyc, iatcp and tcp proteins on downstream regulatory networks in arabidopsis, a microarray analysis was conducted. material for rna extraction was harvested from young inflorescences, comprising flower bud stages before anthesis, of transgenic t plants overexpressing these genes. in total, genes were determined to be differentially regulated following ectopic iatcp expression, the majority being negatively affected. slightly less genes respond to an overexpression of cyc. the lowest number of genes is addressed by ectopic tcp activity.figure numbers of differentially expressed genes caused by ectopic activity of cyc2 transcription factors. the diagram shows the numbers of differentially expressed genes detected with microarrays hybridized with inflorescence cdna from transgenic arabidopsis plants overexpressing either iatcp, cyc or tcp, together with respective floral phenotypes. total target gene numbers are given outside the circles, numbers of target genes solely addressed by respective transcription factors are shown inside the circles. numbers of genes that are mutually addressed by two or all three cyc transcription factors are displayed in intersections. arrows indicate up- or downregulation, asterisks denote mutually addressed genes with a reciprocal expression. scale bars: 2 mm. the cyc transcription factors cyc and iatcp which govern monosymmetry development, address both a larger number of target genes in the heterologous arabidopsis system than tcp this tendency is more obvious regarding only the number of target genes, which are exclusively addressed by a single cyc protein, namely by cyc, by iatcp and only by tcp the capability of the cyc and iatcp genes to address a larger number of downstream targets might reflect their function in the control of monosymmetry formation, even when expressed in a heterologous species forming a polysymmetric corolla such as arabidopsis. target genes are commonly addressed by all three transcription factors and might thus not be linked to specific functions in symmetry regulation. the highest number of overlappingly regulated genes is found between the two crucifer transcription factors, whereas the smallest number of targets is mutually addressed by cyc and tcp. to gain insight into the molecular network acting downstream of iatcp significantly accumulated go terms were detected. for the genes repressed by iatcp the majority of enriched go terms are associated with cell wall-related processes. other enriched go terms are associated with secondary metabolic processes, h2o2catabolic processes and response to red light. additional file summarizes single genes, amongst them cell wall genes that are mutually addressed by ectopic expression of iatcp in arabidopsis inflorescences and by endogenous iatcp in adaxial iberis petals.table enriched go terms in the pool of iatcp1 target genes in arabidopsis inflorescences go term genes ↓ by iatcp1 % arabidopsis infl. genes % fold enrichment listed are go terms that are significantly enriched amongst all genes negatively regulated upon iatcp overexpression in arabidopsis inflorescences in comparison to the arabidopsis inflorescence transcriptome. for each go term, numbers of genes clustering to these categories from the pools of downregulated genes and from the genes forming the arabidopsis inflorescence transcriptome, are given. numbers are also expressed in percent, together with their respective fold enrichment. rna-seq and microarray analyses, both, detected a differential regulation of genes involved in cell wall processes. however, in young transgenic arabidopsis inflorescences all cell wall-related transcripts are downregulated following an ectopic iatcp activity, whereas they are upregulated in older iberis petals after anthesis, in correlation with and presumably also in response to a high endogenous iatcp1-expression. this indicates that the spatial and temporal context of cyc activity is crucial for its proper effect on cell growth and differentiation. discussion monosymmetry in iberis is achieved through differential cell proliferation during late flower development iberis petals are initiated simultaneously and unequal growth begins around the onset of stamen differentiation, from where it proceeds and gains its maximal size difference after anthesis, likely driven by differential cell proliferation. this requires cell division to continue throughout later phases of flower development, which was demonstrated by monitoring the cell division pattern via histone expression from iberis in different petal stages. in older petals, cell division still takes place close to anthesis in both petal types, where it is restricted mainly to the petal tips. similar to iberis, cell division in arabidopsis petals increases throughout flower development until stage and can still be visualized in stage just before anthesis. after anthesis, adaxial iberis petals increase -fold, whereas abaxial petals increase -fold to reach their final size. in contrast, cells of both petal types only double in size from anthesis to maturity. these data indicate that cell division – at least in abaxial petals – proceeds beyond anthesis. transcriptome analysis of petal development of iberis amara rna-seq is being used more and more often as a convenient tool for comparative transcriptome analyses of species with inavailable genome sequences. applying an rna-seq approach, we investigated the iberis transcriptome at the late petal developmental stage a just after anthesis of flowers. comparison of the arabidopsis and iberis petal transcript distribution into functional categories indicates that molecular processes governing late petal development are similar in the two crucifers. interestingly, for the largest group of the iberis petal transcriptome, comprising 31% of the transcripts, no function could be assigned yet. a similar situation with 28% of these genes is observed for arabidopsis. interestingly, comparative analysis of different petal stages in antirrhinum, where flowers form an even more elaborate and complex shaped corolla, revealed that the frequency of the unclassified protein group accounts for over 50% amongst all transcripts expressed in petals. this suggests that a large proportion of genes exert an intriguing, yet uncharacterized, function during petal morphogenesis. analysis of the most abundant transcripts in iberis petal stage a revealed that various enzymes, subsumed in the category misc, are highly expressed in the two petal types, amongst them gdsl-lipases and lipid transfer proteins. both enzyme types have a function in lipid metabolism and have been described as putative target genes of the petal and stamen-specifying b-class mads box transcription factor during petal development in antirrhinum and arabidopsis. the cytochrome p gene cyp76c which is amongst the most highly expressed iberis petal genes, is also strongly expressed in various arabidopsis floral organs, where it participates in the linalool metabolism. linalool, a well-studied attractant for pollinators, is converted further into its oxigenated derivates by cyp76c in autogamous arabidopsis flowers, which likely function in plant defence against pathogens and insect herbivores. two lipoxygenases, lox and lox which exert functions in hormone metabolism, are also highly expressed in iberis petals. the lox protein catalyzes the first dedicated step in jasmonic acid biosynthesis and is activated by the transcription factor tcp in leaves. tcp is also expressed in both iberis petal types, and since lox is localized to chloroplasts, lox2-dependent induction of ja likely takes place in the green petal claws, where ja might exert a negative effect on cell proliferation. the monosymmetric corolla formation of iberis is realized through differential timing of the switch from cell division to cell elongation with almost 15-times more genes upregulated in the small adaxial petals compared to the large abaxial petals, it seems that more genes are required to restrict adaxial petal size during iberis corolla development. the fact that the monosymmetry key regulator iatcp is highly expressed in adaxial petals and represents one of the genes with the largest expression difference implies that iatcp activates a larger number of target genes compared to its inhibitory transcription effects. differential iberis petal development is likely driven by unequal cell division processes. consistently, genes exerting negative effects on cell division are amongst the pool of transcripts upregulated in adaxial petals. in arabidopsis, cytokinin oxidase catalyses the metabolic breakdown of the plant hormone cytokinin and plants with defective ckx genes form flowers with enlarged petals due to an increased cell number. the trihelix transcription factor ptl is expressed in regions of developing arabidopsis flowers, where growth is repressed. ptl expression in the inter-sepal zone is assumed to keep cell proliferation under control to allow the outgrowth of petal primordia. the loss of ptl function results in an increased cell proliferation in that region. petals are initiated in response to localized auxin accumulation, and lampugnani and colleagues speculate this could be evoked via a supportive role of ptl in non-polar auxin efflux through pgps. pgps belong to the superfamily of abc-transporters and function in mediating auxin transport. pgp which is also transcriptionally enriched in adaxial iberis petals, belongs to the same clade as the auxin exporters pgp and pgp, for which double mutant plants show severe growth defects. in addition to transcripts negatively affecting cell proliferation, a large number of genes related to cell wall modifying processes are upregulated in adaxial petals. after an initial phase of organ growth by cell division, final organ size is realised by cell elongation. the primary cell wall of land plants is composed of a strong cellulose microfibril framework cross-linked through xyloglucan chains and embedded in a stabilizing matrix of pectin polysaccharides and structural proteins. plant cell walls provide stability by withstanding tensile stress, and cell growth results from changes in wall extensibility by relenting to this stress. amongst the group of cell wall modifying proteins in adaxial iberis petals are several pectinases, enzymes that function in the de-/methylesterification or degradation of pectin and, by that, might contribute to cell growth via precisely tuning the proportion of pectate in the cell wall. a xyloglucan endotransglucosylase, which cleaves and re-attaches xyloglucan chains in vitro was found to be upregulated in adaxial petals. xet activity correlates with regions undergoing cell expansion, indicating that xets have a role in cell expansion, e.g. through the biogenesis of newly formed cell walls. in addition, several iberis glycosyl hydrolases were upregulated in adaxial petals, which form a diverse group of enzymes that hydrolyze glycosidic bonds. one arabidopsis homolog of the ghs belongs to the subfamily of the endo- -ß-glucanases korrigan, which is essential for cell elongation and cellulose synthesis in arabidopsis hypocotyls. several transcripts encoding for a cobra-like protein, two proline rich extension-like receptor kinases, an arabinogalactan protein and a formin are upregulated in adaxial petals and might act as a potential connection between the cytoplasma and cell wall. these proteins are, thus, part of the molecular continuum that connects the cytoskeletal network, the cytoplasm and the cell wall, and which contributes to cell expansion control in arabidopsis. arabidopsis homologs of several of the floral cell wall-related genes, such as a polygalacturonase, a xet and a pectin lyase like member are also predominately expressed in the cell elongation zone of root tips and only weakly in the preceeding meristematic region, where growth is realized by cell divisions. this supports their general function in cell wall modification, both in a floral and root context, contributing to tissue growth by mediating cell elongation processes. the determination of final organ size requires the precise coordination of both, cell division and cell elongation processes, and likely depends on a tight cellular communication. amongst all adaxial-petal specific genes, a group of transcripts involved in cell-cell signalling is significantly enriched. these ralfs are small peptides, which are important for many developmental processes and regarded as novel plant hormones. in grass leaves, ralfs were found to be mainly expressed in young elongating cells beyond the meristematic zone, and expression is almost absent in mature cells. as ralfs are thought to exert a negative effect on cell growth, their expression strength and location is likely precisely adjusted during organ development to achieve an accurate guidance of cell expansion without completely blocking it. summing up, a significantly increased number of transcripts involved in cell wall modification and cell-cell signalling, together with genes regulating the restriction of cell division and auxin transport were detected in the pool of transcripts enriched in small, adaxial iberis petals. hence, unequal petal growth is likely achieved via an earlier stop of cell division in adaxial petals with a subsequent earlier onset of cell expansion, whilst abaxial petal cells continue dividing. in iberis, cell division ceases in an acropetal gradient, where cells at the tip keep on dividing longest, similar as in other plant species. thus, an unequal stop of cell division might be realised through a faster acropetal progression of a cell division stop front in adaxial petals compared to abaxial petals. ectopic expression of iatcp causes expression changes of cell wall related genes in arabidopsis microarray analyses aimed at comparing the molecular networks that are addressed in inflorescences by iatcp and tcp causing smaller petal formation, and by cyc, giving rise to the development of larger petals. a considerable number of genes is addressed by all three cyc proteins together. these might be transcripts that constitute a common basal set of tcp/cyc transcription factor targets genes in arabidopsis thaliana. interestingly, in the heterologous arabidopsis system, more genes are exclusively addressed by iatcp from iberis and cyc from antirrhinum, developing a monosymmetric flower, than by tcp from arabidopsis, forming a polysymmetric corolla. these quantitative differences indicate that iatcp and cyc might regulate more target genes, implying that a larger downstream regulatory network realizes monosymmetric compared to polysymmetric corolla development. therefore, in addition to cis-regulatory changes that led to a heterochronic cyc gene expression shift in the brassicaceae, promoting corolla monosymmetry evolution, also a divergence of trans-acting cyc proteins likely contributed to the establishment of this morphological novelty. the largest overlap in target gene number is shared between iatcp and tcp these genes comprise several transcripts involved in cell wall-related and signalling processes and the activity of these exclusive crucifer targets could be essential for the petal size restriction in arabidopsis, as only transgenic plants ectopically expressing a crucifer cyc protein develop flowers with smaller petals. amongst the population of target genes downregulated in response to heterologous iatcp expression in arabidopsis, again a high proportion is involved in cell wall modification processes. this indicates that iatcp addresses these genes in arabidopsis and that the high abundance of this gene class detected with rna-seq in adaxial iberis petals is the effect of the strong iatcp expression there. while cell wall genes are positively affected in older adaxial iberis petals, where iatcp expression is strongly enhanced, iatcp activity has a negative effect on cell wall genes in young arabidopsis inflorescences overexpressing iatcp tcp transcription factors are known to exert either an activating or repressive effect on organ growth, depending on their tissue specific expression. as tcp proteins act as dimers, the outcome of their activity might differ in dependence of available interaction partners in different organs or developmental stages. similar development-dependent regulatory effects were shown previously for the knox/bel transcription factor bellringer, which interacts with other bel and knox proteins. one of its targets, the pectin methylesterase pme is repressed by blr in the shoot meristem, to allow coordinated formation of primordia, but is activated by blr in internodes, where it promotes cell elongation.  an rna-seq expression analysis on adaxial and abaxial stage a iberis petals reveals a significantly increased number of cell wall-related transcripts in adaxial petals. this indicates that differential petal growth, a characteristic of the monosymmetric iberis corolla, is achieved through an earlier stop of the cell proliferation phase followed by the onset of cell expansion and differentiation in the smaller adaxial petals, while abaxial petal cells likely still continue growth via cell division. as ectopic expression of iatcp in arabidopsis addresses cell wall-related genes, the enhanced occurrence of this gene class in adaxial iberis petals is very likely also a direct consequence of the strong iatcp expression there. moreover, crucifer monosymmetry evolution likely relied on the combination of cis-regulatory changes, leading to a late adaxial iatcp expression and also on trans-regulatory changes mediated by differences in the cyc proteins themselves, altering the set of potential target genes that contribute to the large petal size differences in iberis amara. 
furthermore, microarrays were used to detect and compare quantitative differences in tcp target genes in transgenic arabidopsis plants ectopically expressing different tcp transcription factors. about gb of sequence data was generated, processed and re-assembled into likely iberis unigenes, from which showed high sequence homology to arabidopsis proteins. in addition, it was used as a scaffold to detect genes differentially expressed between the small, adaxial and the large, abaxial petals in order to understand the molecular mechanisms driving unequal petal growth. the increased occurrences of genes implicated in cell wall modification and signalling implies that unequal petal growth is achieved through an earlier stop of the cell proliferation phase in the small, adaxial petals, followed by the onset of cell expansion. in the co-evolution between insects and plants, the establishment of floral monosymmetry was an important step in angiosperm development as it facilitated the interaction with insect pollinators and, by that, likely enhanced angiosperm diversification. in antirrhinum majus, the tcp transcription factor cycloidea is the molecular key regulator driving the formation of floral monosymmetry. the transcriptome gives detailed insight into the molecular mechanisms governing late petal development. although most brassicaceae form a polysymmetric corolla, six genera develop monosymmetric flowers with two petal pairs of unequal size. in the monosymmetric crucifer iberis amara, formation of the different petal pairs coincides with a stronger expression of the cyc-homolog iatcp in the small, adaxial petals. amongst all genes upregulated in adaxial petals, a significantly enhanced proportion is associated with cell wall modification and cell-cell signalling processes. this process, which forms the monosymmetric corolla of iberis amara, is likely driven by the enhanced activity of iatcp in adaxial petals. in this study, rna-seq was employed to reconstruct the petal transcriptome of the non-model species iberis amara. far more genes are expressed in adaxial compared to abaxial petals implying that iatcp activates more genes than it represses.
 in the co-evolution between insects and plants, the establishment of floral monosymmetry was an important step in angiosperm development as it facilitated the interaction with insect pollinators and, by that, likely enhanced angiosperm diversification. in antirrhinum majus, the tcp transcription factor cycloidea is the molecular key regulator driving the formation of floral monosymmetry. although most brassicaceae form a polysymmetric corolla, six genera develop monosymmetric flowers with two petal pairs of unequal size. in the monosymmetric crucifer iberis amara, formation of the different petal pairs coincides with a stronger expression of the cyc-homolog iatcp in the small, adaxial petals.  in this study, rna-seq was employed to reconstruct the petal transcriptome of the non-model species iberis amara. about gb of sequence data was generated, processed and re-assembled into likely iberis unigenes, from which showed high sequence homology to arabidopsis proteins. the transcriptome gives detailed insight into the molecular mechanisms governing late petal development. in addition, it was used as a scaffold to detect genes differentially expressed between the small, adaxial and the large, abaxial petals in order to understand the molecular mechanisms driving unequal petal growth. far more genes are expressed in adaxial compared to abaxial petals implying that iatcp activates more genes than it represses. amongst all genes upregulated in adaxial petals, a significantly enhanced proportion is associated with cell wall modification and cell-cell signalling processes. furthermore, microarrays were used to detect and compare quantitative differences in tcp target genes in transgenic arabidopsis plants ectopically expressing different tcp transcription factors.  the increased occurrences of genes implicated in cell wall modification and signalling implies that unequal petal growth is achieved through an earlier stop of the cell proliferation phase in the small, adaxial petals, followed by the onset of cell expansion. this process, which forms the monosymmetric corolla of iberis amara, is likely driven by the enhanced activity of iatcp in adaxial petals. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords brassicaceaemonosymmetrycyctcp1rna-seqmicroarrayissue-copyright-statement© the author 2014 