Flavonoid metabolons (weakly-bound multi-enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein–protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS)
FNSII/chalcone isomerase (CHI)
FNSII/dihydroflavonol 4-reductase (DFR)
CHS/CHI
CHI/DFR
and flavonoid 3′-hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage-dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co-suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long-suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.