Selective accumulation of delphinidin derivatives in tobacco using a putative flavonoid 3',5'-hydroxylase cDNA from Campanula medium

Blue flowers generally contain 3',5'-hydroxylated anthocyanins (delphinidin derivatives) as pigments, which are formed only in the presence of flavonoid 3',5'-hydroxylases (F3'5'H). Heterologous expression of a F3'5'H gene therefore provides an opportunity to produce novel blue flowers for a number of ornamental plants missing blue flowering varieties. However, our previous study indicated difficulties in obtaining good accumulation of delphinidin derivatives in plants expressing F3'5'H. Here we report the isolation of a putative F3'5'H cDNA (Ka1) from canterbury bells (Campanula medium) and its expression in tobacco. Surprisingly, compared with other F3'5'H cDNAs, Ka1 encoded a protein with a unique primary structure that conferred high competence in the accumulation of delphinidin derivatives (up to 99% of total anthocyanins) and produced novel purple flowers. These results suggest that, among F3'5' H cDNAs, Ka1 is the best genetic resource for the creation of fine blue flowers by genetic engineering.     

Genetic engineering of the anthocyanin biosynthetic pathway with flavonoid-3′,5′-hydroxylase: specific switching of the pathway in petunia

Flavonoid-3',5'-hydroxylase (F3'5'H) is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, which are generally required for the expression of blue or purple flower color. It has been predicted that the introduction of this enzyme into a plant species that lacks it would enable the production of blue or purple flowers by altering the anthocyanin composition. We present here the results of the genetic engineering of petunia flower color, pigmentation patterns and anthocyanin composition with sense or antisense constructs of the F3'5'H gene under the control of the CaMV 35S promoter. When sense constructs were introduced into pink flower varieties that are deficient in the enzyme, transgenic plants showed flower color changes from pink to magenta along with changes in anthocyanin composition. Some transgenic plants showed novel pigmentation patterns, e.g. a star-shaped pattern. When sense constructs were introduced into blue flower petunia varieties, the flower color of the transgenic plants changed from deep blue to pale blue or even pale pink. Pigment composition analysis of the transgenic plants suggested that the F3'5'H transgene not only created or inhibited the biosynthetic pathway to 3',5'-hydroxylated anthocyanins but switched the pathway to 3',5'-hydroxylated or 3'-hydroxylated anthocyanins.     

A Novel Method to Clone P450s with Modified Single-Specific-Primer PCR

We present a method to identify cDNA clones of a cytochrome P450 enzyme. Flavonoid-3', 5'-Hydroxylase (F3',5'H), the key enzyme for the expression of blue or purple color in flowers, was cloned as an example. We have made a catalog of cDNA fragments encoding conserved regions of P450s for petunia (Petunia hybrida Vilm.) petals. Single specific primers were designed for these cDNA sequences and RT-PCRs were performed with cDNA templates. The amplified bands were tested for linkage to the delphinidin producing phenotype using a backcrossed population that had been prepared to have a genetic background of cyanidin-type petunia but segregated for the hydroxylation at the B-ring of anthocyanin. We were successful in amplifying a cDNA fragment that has close linkage to the F3',5'H gene. A full length cDNA clone of the F3',5'H gene was isolated using the amplified fragment as a probe.