SUMMARYBecause structural modifications of flavonoids are closely related to their properties, such as stability, solubility, flavor and coloration, characterizing the enzymes that catalyze the modification reactions can be useful for engineering agriculturally beneficial traits of flavonoids. In this work, we examined the enzymes involved in the modification pathway of highly glycosylated and acylated anthocyanins that accumulate in Lobelia erinus. Cultivar Aqua Blue (AB) of L. erinus is blue-flowered and accumulates delphinidin 3-O-p-coumaroylrutinoside-5-O-malonylglucoside-3 0 5 0 -O-dihydroxycinnamoylglucoside (lobelinins) in its petals. Cultivar Aqua Lavender (AL) is mauve-flowered, and LC-MS analyses showed that AL accumulated delphinidin 3-O-glucoside (Dp3G), which was not further modified toward lobelinins. A crude protein assay showed that modification processes of lobelinin were carried out in a specific order, and there was no difference between AB and AL in modification reactions after rhamnosylation of Dp3G, indicating that the lack of highly modified anthocyanins in AL resulted from a single mutation of rhamnosyltransferase catalyzing the rhamnosylation of Dp3G. We cloned rhamnosyltransferase genes (RTs) from AB and confirmed their UDP-rhamnose-dependent rhamnosyltransferase activities on Dp3G using recombinant proteins. In contrast, the RT gene in AL had a 5-bp nucleotide deletion, resulting in a truncated polypeptide without the plant secondary product glycosyltransferase box. In a complementation test, AL that was transformed with the RT gene from AB produced blue flowers. These results suggest that rhamnosylation is an essential process for lobelinin synthesis, and thus the expression of RT has a great impact on the flower color and is necessary for the blue color of Lobelia flowers.