“The usual suspects”- analysis of transcriptome sequences reveals deviating B gene activity in C. vulgaris bud bloomers
© Behrend et al.; licensee BioMed Central. 2015
Received: 5 November 2014
Accepted: 23 December 2014
Published: 21 January 2015
The production of heather (Calluna vulgaris) in Germany is highly dependent on cultivars with mutated flower morphology, the so-called diplocalyx bud bloomers. So far, this unique flower type of C. vulgaris has not been reported in any other plant species. The flowers are characterised by an extremely extended flower attractiveness, since the flower buds remain closed throughout the complete flowering season. The flowers of C. vulgaris bud bloomers are male sterile, because the stamens are absent. Furthermore, petals are converted into sepals. Therefore the diplocalyx bud bloomer flowers consist of two whorls of sepals directly followed by the gynoecium.
A broad comparison was undertaken to identify genes differentially expressed in the bud flowering phenotype and in the wild type of C. vulgaris. Transcriptome sequence reads were generated using 454 sequencing of two flower type specific cDNA libraries. In total, 360,000 sequence reads were obtained, assembled to 12,200 contigs, functionally mapped, and annotated. Transcript abundances were compared and 365 differentially expressed genes detected. Among these differentially expressed genes, Calluna vulgaris PISTILLATA (CvPI) which is the orthologue of the Arabidopsis B gene PISTILLATA (PI) was considered as the most promising candidate gene. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT PCR) was performed to analyse the gene expression levels of two C. vulgaris B genes CvPI and Calluna vulgaris APETALA 3 (CvAP3) in both flower types. CvAP3 which is the orthologue of the Arabidopsis B gene APETALA 3 (AP3) turned out to be ectopically expressed in sepals of wild type and bud bloomer flowers. CvPI expression was proven to be reduced in the bud blooming flowers.
Differential expression patterns of the B-class genes CvAP3 and CvPI were identified to cause the characteristic morphology of C. vulgaris flowers leading to the following hypotheses: ectopic expression of CvAP3 is a convincing explanation for the formation of a completely petaloid perianth in both flower types. In C. vulgaris, CvPI is essential for determination of petal and stamen identity. The characteristic transition of petals into sepals potentially depends on the observed deficiency of CvPI and CvAP3 expression in bud blooming flowers.
Keywords454 sequencing Bud flowering Floral organ identity Heather Homeotic mutant Real-time PCR Transcriptome Transcription factor
Calluna vulgaris (Ericaceae) is an important ornamental crop for autumn planting in Northern Europe. The demand for C. vulgaris has constantly been increasing during the last years because of the longevity of a special mutant in flower morphology, the so-called bud bloomers. Today, 80% of all protected varieties of C. vulgaris in Germany are bud bloomers  and make C. vulgaris one of the top selling landscaping plants in Germany . The bud bloomers show an unique flower architecture with combination of unopened flowers and absence of any organ development in whorl III: the perianth of bud bloomers remains closed during the whole flowering period, stamens are missing and petals are converted into sepals . Bud blooming individuals were found in natural populations in 1936 and 1948 in Great Britain as well as in 1970 in the Netherlands  and were introduced as commercial varieties. Due to the shielding from cross-pollination by closed perianth organs and the impossibility of self-pollination due to the loss of stamens and the presence of a second whorl of robust sepals instead of softer petals, the flower buds of bud bloomers display a prolonged flower attractiveness compared to other flower types of C. vulgaris. The extended longevity of flowers is a highly desired trait promoting the bud bloomers’ economic success compared to varieties with wild type or filled flowers. An attractive flower morphology is one of the major selection targets in ornamental breeding.
The genetics of different flower architectures can be explained by the ABC model of floral organ identity and its variants. It describes the interaction of the homeotic transcription factors in determination of floral organ identity [7-9]. In the classical ABC model, the expression of A genes is responsible for development of sepals in whorl I, activity of B genes in combination with C genes is necessary to determine organ identity of stamens in whorl III. B gene together with A gene function induces the formation of petals in whorl II. Finally, C gene expression on its own defines carpels. Since B genes in combination with A und C are responsible for the determination of organs in whorl II and III, and these organs are affected in both bud bloomer mutants, a deficiency in B gene function is the most convincing hypothesis for formation of the bud flowering phenotype in C. vulgaris. Accordingly, the polystyla bud blooming type corresponds perfectly to the phenotype of a classical B gene mutant as described in Arabidopsis thaliana (thale cress) , Antirrhinum majus (snapdragon) [11,12], and several other plant species [13-25]. The closed perianth in bud bloomers is probably the result of petal loss, as studies in Arabidopsis B gene mutants show .
In first gene expression analyses, Borchert et al. (2009)  already found a reduced expression of the B gene CvAP3 in floral organs of whorl II in three diplocalyx bud flowering cultivars indicating the presence of a second whorl of sepals instead of petals which is expected according to the model. On the other hand, the formation of petaloid sepals in all flower types of C. vulgaris points to an ectopic expression of B genes in whorl I, resulting in conflicting hypotheses with regard to the genetics of the diplocalyx bud flower type.
Therefore, the aim of the current study was to compare the transcriptome of the wild type (wt) and the diplocalyx bud bloomer flowers (bud) of C. vulgaris and to deduce a hypothesis for the genetic basis of the diplocalyx bud bloomer flower architecture.
454 sequencing and assembly
Overview on 454 data
Total read number in contigs
Average length contigs (nt)
Average length isotigs (nt)
Average length singeltons (nt)
Annotation of sequences
Number of contigs in each library during processing in blast2go
Without BLAST hit
With BLAST result
With mapping result
Differential gene expression
Functional classification of differentially expressed genes
171 differentially expressed genes (46.8%) did not match homologues proteins in the data base. Differentially expressed genes that could be annotated were checked for functional classification in biological processes to identify reasonable candidates for the bud flowering phenotype. GO enrichment analysis pointed out to overrepresentation of GO terms related to ribosome function in wt. In addition, the data sets of differentially expressed genes in the GO categories “flower development”, “floral whorls development” and “sequence specific DNA binding transcription factor activity” were carefully checked for probable candidate genes (Additional file 3). The following annotated contigs were assigned to flower or floral whorl development: DNAj, glycerol-3-phosphate acyltransferase, 26S proteasome non ATPase regulatory subunit rpu 12a, basic blue protein, 3-ketoacyl-synthase 6. None of these was considered as potential candidate gene for the bud flowering phenotype. In addition, the BLAST and mapping results of four differentially expressed transcription factors were monitored. Two putative transcription factors, a GAGA binding transcriptional activator and an ethylene responsive transcription factor RAP2-3, are considered to be involved in stress response. A putative E2FE like transcription factor is involved in cell proliferation. Consequently, these three genes were also excluded as candidate genes. The fourth one, contig07420 which exerts a homology to PISTILLATA (PI), belonging to the class B genes, of Actinidia chinensis (yellow kiwi fruit), was identified as a promising candidate gene and was named CvPI.
The genes differentially expressed in the different flower type of C. vulgaris were also compared to a list of differentially expressed genes in Arabidopsis B gene mutants from microarray studies . 51 of the contigs from C. vulgaris could be assigned to counterparts in the Arabidopsis data set, at least on protein family level. Most matches (20) were obtained with the pi-1 mutant. 16 matches were found with ap3-1 mutant and 15 with the ap3-3 mutant. 45 C. vulgaris contigs showed a similar expression pattern as the corresponding genes in Arabidopsis in at least at one of three time points monitored in the Arabidopsis study (Additional file 2).
Evaluation of candidate gene by real-time PCR (qRT PCR)
In C. vulgaris bud bloomers of the diplocalyx type male flower organs are missing (Figure 1B), petals are converted into a second whorl of sepals and the flower remains closed. This flower type is a highly desired trait in ornamental plant breeding, since the bud bloomers’ flowers have an extended flowering period. The aims of this study were to characterise the gene expression profile of C. vulgaris diplocalyx bud bloomers by a broad transcriptome study and deduce candidate genes causing the diplocalyx bud flowering phenotype by the comparison with wt flowers.
Unopened flowers which later drop and form no siliques have been described in Arabidopsis LSU4 mutants . In addition, flowers of pi-1 mutants as well as transgenic Arabidopsis plants ectopically expressing LMADS8 or LMADS9 flowers were termed as unopened . The morphology of these mutants points to a crosslink of floral organ morphology and flower opening [26,33]. This circumstance is a good explanation for the bud bloomers’ phenotype in C. vulgaris. Since stamen development was not detectable in the diplocalyx type  or stamens have carpel-like character in the polystyla type and petals are replaced by sepals, organs responsible for flower opening are missing in these flower types. The identity of the affected organs points to a modified expression of a B gene in the bud flowering phenotype, since stamens and petals are the mutated organs. The apparent absence of third whorl organs may reflect their complete incorporation into the fourth whorl gynoecium . Upstream regulators of B gene expression as UFO, LEAFY or AP1 are unlikely to be affected in the C. vulgaris bud bloomer mutants, because dysfunctions in these genes would cause severe flower malformations: UFO mutants in Arabidopsis display filamentous structures instead of flowers . LEAFY mutants produce leafs and associated lateral shoots instead of early flowers, later developing flowers are substituted by structures with flower and leaf traits [36,37]. In AP1 mutants of Arabidopsis, sepals are replaced by bracts, petals are missing and additional flowers arise in the axils of the first whorl organs [38,39].
However, in model plants, typical B gene loss of function mutants display a second whorl of sepals instead of petals and the formation of carpeloid stamens. In Arabidopsis, the B genes APETALA3 (AP3) and PISTILLATA (PI) are responsible for the control of organ identity in whorl II (petals) and III (stamens) . Since AP3 and PI function as a heterodimer in Arabidopsis, mutations of either AP3 or PI cause identical phenotypes with altered organ identity in whorl II and whorl III . The function of the B class genes AP3 and PI seems to be highly conserved during evolution of flowering plants. Because C. vulgaris bud bloomers phenotype shows conflicting characters compared to a classical B gene mutant - on the one hand petaliod sepals, on the other hand loss of stamens and petals - a broad RNA sequencing approach was chosen to find genes differentially expressed in wt and the diplocalyx bud flowering phenotypes of C. vulgaris. These data have been compared to the data set of Wuest et al. 2012  to elucidate parallels and differences with Arabidopsis B gene mutants.
High throughput 454 sequencing was found to be an effective method to characterise the transcriptomes of different flower types of C. vulgaris. Next generation sequencing is the state of the art approach for broad gene expression analysis relative to methods such as microarrays and subtractive cDNA libraries [40-42]. The 454 sequencing technology is an effective tool for tissue specific functional genomics in non-sequenced plants species, because it is capable to capture also rarely expressed transcripts as transcription factors [43-49] and delivers massive numbers of additional transcript sequences which were useful in the presented study for qRT PCR normalizer choice. In addition, the obtained data bases of C. vulgaris floral transcriptomes are valuable resources for further research on flower related traits in this ornamental crop.
From the set of 365 differentially expressed genes, CvPI was considered to be the most plausible candidate responsible for causing the diplocalyx flower mutant. Moreover, a significantly reduced expression level of CvPI in diplocalyx bud bloomers has been confirmed by qRT-PCR. Nevertheless, the lack of CvPI expression in C. vulgaris bud bloomers is not causing the typical phenotype of a B gene mutant as anticipated from Arabidopsis, since in diploxcalys flower mutants, stamens are completely missing. A similar phenotype has been found in a peloric mutant of Phalaenopsis equestris in which the development of stamens and staminodes was completely eliminated  and the expression of the B gene PeMADS5 was not detectable in the floral tissue.
In C. vulgaris, the expression of CvPI was found to be high in petals and stamens of the wild type as expected from the ABC model. In contrast, CvAP3 expression was prominent in whorl I-III of wt and diplocalyx bud blooming flowers. In opposite to CvAP3, hardly any CvPI transcript was detectable in the floral tissues of the bud flowering plants by qRT PCR. According to the classical ABC model and its modifications, the expression of the AP3-like gene is restricted to whorls II and III . CvAP3 expression in whorl I is considered to cause the petaloid character of C. vulgaris sepals in both studied flower types. This finding is supported by earlier expression analysis in C. vulgaris  and data from multiple species, including important floriculture crops as Tulipa gesneriana (garden tulip) , Lilium longiflorum (Easter lily) , and Agapanthus praecox (common agapanthus) . Since CvPI expression is absent from floral tissue of diplocalyx bud bloomers, it is assumed that petal and stamen development in C. vulgaris depends on the binding of CvPI in a regulatory complex of MADS box genes containing CvAP3 and the absence of CvPI is causing to the development of a second whorl of petaloid sepals and the absence of stamens. Due to the petaloid character of this extra whorl of sepals and the expression level of CvAP3 in whorls II and III, it is concluded that only the lack of CvPI expression is causing the altered flower architecture and not a combined dysfunction of CvPI and CvAP3. In addition, the finding of CvAP3 transcripts in carpels of diplocalyx bud bloomers without stamen character also points to the hypothesis of an exclusive dysfunction of CvPI being responsible for the loss of stamens.
To elucidate the consequences of putative CvPI dysfunction in C. vulgaris the list of differentially expressed genes in young flowers of C. vulgaris comparing the diplocalyx bud bloomer and wild type flowers was compared to published data from Arabidopsis B gene mutants . In this study 2100 genes were identified to be differentially regulated in B gene mutants. In Arabidopsis pi1-1 mutants, GO terms like petal development, stamen development, floral organ formation, floral organ morphogenesis, and regulation transcription were found to be significantly enriched. In contrast, these GO terms were not enriched in the C. vulgaris data set. The major difficulty in functional analysis of differentially expressed genes in C. vulgaris bud bloomers proved to be the low informative value of GO term enrichment analysis. The annotation of C. vulgaris sequences did not identify single genes but gene families or only protein motives, making obtained GO terms rather unspecific. This is attributed to the low sequence identity between C. vulgaris and model plants and to the incomplete annotation of sequence data from closer relatives. Therefore, for more detailed results using GO analysis of the present 454 read data, more detailed sequence information of C. vulgaris or close relatives is needed.
Further studies on the bud bloomers phenotype in C. vulgaris are planned including comparison of B gene expression in the diplocalyx and polystyla type and the localisation of transcripts with an in situ hybridisation approach to unveil CvPI and CvAP3 expression pattern during floral development. Protein and DNA binding studies with CvPI and CvAP3 protein from bud bloomer and wild type genotypes are necessary to clarify the composition and function of homeotic floral MADS box protein complexes in C. vulgaris flower development. Of special interest in C. vulgaris is the investigation of the crosslink of B gene expression and the genetic regulation of carpel development reported from Arabidopsis , since several cultivars with bud flowering phenotype suffer from carpel malformation . Moreover, mapping of CvPI expression in an existing mapping population  is planned to check the cosegregation with the trait flower type.
The B genes CvPI und CvAP3 have been found to play crucial roles in the development to the diplocalyx bud bloomer mutants of C. vulgaris, which are of major economic significance in this important landscaping plant. Ectopic expression of CvAP3 in sepals seems to be responsible for their petaloid character. A drastically reduced expression of CvPI in flowers of diplocalyx bud bloomer mutants points to a central role of this transcription factor in the formation of this flower type. Further research is necessary to figure out the differences in B gene expression between polystyla and diplocalyx bud bloomers in C. vulgaris.
Plants of bud flowering varieties (‘Maria’ , ‘Anett’ , ‘Marlis’ , ‘Ginkel’s Glorie’) and genotypes with wild type flowers (‘Boskoop’ , ‘Hammonidii’ , F1, Niederohe) were kept in the IGZ greenhouse in winter and under field conditions in frost free periods. ‘Maria’ , ‘Anett’ , ‘Marlis’ , ‘Ginkel’s Glorie’ , Boskoop’ , ‘Hammonidii’ are commercially available varieties. The wild type Niederohe was grown from plant material collected in Germany. The genotype F1 originated from the cross ‘Maria’ x ’Boskoop’. Flowers from all genotypes were collected and dissected into bracts, sepals, petals, stamens (if present) and carpels. Floral organs and leaves were conserved in RNAlater (Invitrogen) and stored at −80°C.
RNA extraction and cDNA synthesis
Total RNA was extracted with the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions with modifications as published in Dhanaraj et al. (2004)  including intensively on column washing with 80% EtOH. The complete digestion of genomic DNA was performed using TurboDNase (Ambion) according to the manufacturer’s protocol. RNA was quantified using the Nanodrop spectrometer (Thermo Scientific). First strand cDNA synthesis was carried out using the QuantiTec Reverse Transcription Kit (Qiagen). Resulting cDNA concentrations were determined with a Qubit Fluorimeter (Invitrogen).
Library construction and 454 sequencing
Construction of two tagged (TCTACT bud/TGTATC wt) 3’-fragment cDNA libraries from C. vulgaris flower tissue of the bud bloomer ‘Maria’ and its wild type flowering offspring F1 and subsequent 454 sequencing was performed by vertis Biotechnolgie AG, Freising, Germany. Quality checked and adapter trimmed sequences were obtained in fastq format sorted according to the sequence tag. Obtained fastq files were split into fasta and qual files with MIRA 3.0.5  by the convert_project command. For expression analysis, sequences from plastids, endophytes, mitochondria, and for rRNA, were removed using SeqClean .
Sequence annotation, read number determination and expression analysis
Sequence reads were assembled and mapped using the cDNA option of GS DeNovoAssembler (Newbler) 2.5.3 (Roche). BLAST search (blastx, NCBI nr, 1.0 E-3), mapping and annotation (default options) was performed in blast2go . Three transcriptome data bases were obtained: two tag-sorted specific for one flower type each, and a common one containing both libraries (backbone). For in silico expression analysis, transcript abundances were obtained by mapping the flower type specific reads to the common backbone. Only contigs containing more than two reads were used in transcript profiling. Differentially expressed contigs were identified using the Audic Claverie algorithm  (p = 0.01, Bonferroni correction) with the web tool IDEG6 .
qRT PCR analysis
qRT PCR primers designed for amplification of products from 80-120 bp
Product size in bp
CvDisease resistance protein [contig02315]
CvTATA binding [contig05402]
Cv18S rRNA, [GenBank: AF419791]
Availability of supporting data
The raw sequence reads and the result table from the in silico expression analysis have been deposited at NCBI Gene Expression Omnibus (GEO) database under the accession GSE60105. The transcriptome shotgun assembly projects have been deposited at NCBI GenBank under the GenBank accessions GBSW00000000 (backbone) and GBRS00000000 (flower type specific). The versions described in this paper are the first versions, GBSW01000000 and GBRS01000000.
The BLE (Federal Office of Agriculture and Food, Germany) on behalf of the German Federal Ministry of Food, Agriculture and Consumer Protection (BMELV) (support code: 511–06.01-28-1-43.038-07) provided financial support. We thank Janett Grimmer and Katja Krueger for technical assistance. Joerg Krueger is acknowledged for providing IT resources for data analysis. Ralph Heinrich and Magdalena Stock gave helpful hints on data analysis. We thank Rosa Herbst for proofreading.
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