- Petal spots are widespread in angiosperms and are often implicated in pollinator attraction. Clarkia gracilis petals each have a single red-purple spot that contrasts against a pink background. The position and presence of spots in C. gracilis are determined by the epistatic interaction of alleles at two as yet unidentified loci.
- We used HPLC to identify the different pigments produced in the petals, and qualitative and quantitative RT-PCR to assay for spatio-temporal patterns of expression of different anthocyanin pathway genes.
- We found that spots contain different pigments from the remainder of the petal, being composed of cyanidin/peonidin-based, instead of malvidin-based anthocyanins. Expression assays of anthocyanin pathway genes showed that the dihydroflavonol-4-reductase 2 (Dfr2) gene has a spot-specific expression pattern and acts as a switch for spot production. Co-segregation analyses implicated the gene products of the P and I loci as trans-regulators of this switch. Spot pigments appear earlier in development as a result of early expression of Dfr2 and the flavonoid 3′ hydroxylase 1 (F3′h1) gene. Pigments in the background appear later, as a result of later expression of Dfr1 and the flavonoid 3′-5′ hydroxylase 1 (F3′5′h1) genes.
- The evolution of this spot production mechanism appears to have been facilitated by duplication of the Dfr gene and to have required substantial reworking of the anthocyanin pathway regulatory network.