"The usual suspects"- analysis of transcriptome sequences reveals deviating B gene activity in C. vulgaris bud bloomers

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

C. vulgaris flower types. Flowers of C. vulgaris, A - wild type flower with leaves (L), flower organs to the centre: bracts (Br), sepals (Ca: calyx), petals (Co: corolla), stamens (A: androecium), and carpels (G: gynoecium), B - bud bloomer’s flower, diplocalyx type, flower organs to the centre: bracts (Br), sepals (Ca), sepals (Ca), and carpels (G), C - bud bloomer’s flower, polystyla type, cultivar ‘David Eason’, flower organs to the centre: bracts (Br), sepals (Ca), sepals (Ca), carpeloid stamens (G), carpels (G).

Figure 2

Contig length. Distribution of contig lengths after assembly of all 454 sequences reads.

Figure 3

Functional annotations based on GO categories of contigs from wt library, bud library assembly, and contigs differently expressed in C. vulgaris bud bloomer and wild type flowers. BP – biological process, MF – molecular function, CC – cellular component.

Figure 4

Differential GO term distribution among differentially expressed genes. GO term enrichment analysis by Fischer’s exact text.

Figure 5

Expression pattern of CvPI . Normalised gene expression (five reference genes) in the bud blooming phenotype and the wild type shown as fold change (2^-ΔΔCt) of arbitrary units compared the reference tissue of F1 (wt).

Figure 6

Expression pattern of CvAP3. Normalised gene expression (five reference genes) in the bud blooming phenotype and the wild type shown as fold change (2^-ΔΔCt) of arbitrary units compared the reference tissue of F1 (wt).