Many plant species exhibit a reduced range of flower colors due to the lack of an essential gene or to the substrate specificity of a biosynthetic enzyme. Petunia does not produce orange flowers because dihydroflavonol 4-reductase (DFR) from this species, an enzyme involved in anthocyanin biosynthesis, inefficiently reduces dihydrokaempferol, the precursor to orange pelargonidin-type anthocyanins. The substrate specificity of DFR, however, has not been investigated at the molecular level. By analyzing chimeric DFRs of Petunia and Gerbera, we identified a region that determines the substrate specificity of DFR. Furthermore, by changing a single amino acid in this presumed substrate-binding region, we developed a DFR enzyme that preferentially reduces dihydrokaempferol. Our results imply that the substrate specificity of DFR can be altered by minor changes in DFR.

SummarySome angiosperms are limited to a range of possiblē ower colors. This limitation can be due to the lack of an anthocyanin biosynthetic gene or to the substrate speci®city of a key anthocyanin biosynthetic enzyme, dihydro¯avonol 4-reductase (DFR). Cymbidium hybrida orchid¯owers primarily produce cyanidin-type (pink to red) anthocyanins and lack the pelargonidin-type (orange to brick-red) anthocyanins. To investigate the underlying molecular mechanism of this¯ower color range, we cloned a Cymbidium DFR gene and transformed it into a DFR ± petunia line. We found that the Cymbidium DFR did not ef®ciently reduce dihydrokaempferol (DHK), which is an essential step for pelargonidin production. Phylogenetic analysis of a number of DFR sequences indicate that the inability to catalyze DHK reduction has occurred at least twice during angiosperm evolution. Our results indicate that developing a pelargonidin-type orange¯ower color in Cymbidium may require the transformation of a DFR gene that can ef®ciently catalyze DHK reduction.

HighlightReducing the function of PAL, the first enzyme in the phenylpropanoid pathway, in Brachypodium distachyon alters cell wall composition, increases fungal susceptibility, but minimally affects caterpillar herbivory and abiotic stress tolerance.