Naonobu Noda

Crystal structure of UDP‐glucose:anthocyanidin 3‐O‐glucosyltransferase from Clitoria ternatea

The anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea (Ct3GT-A) was expressed in Escherichia coli, and the three-dimensional structure of Ct3GT-A was determined using X-ray crystallography. This report describes the architecture of Ct3GT-A, including the structures of the donor- and acceptor-binding sites.

Structural basis for acceptor-substrate recognition of UDP-glucose: anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea

UDP‐glucose: anthocyanidin 3‐O‐glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP‐glucose to anthocyanidins such as delphinidin. After the acylation of the 3‐O‐glucosyl residue, the 3′‐ and 5′‐hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor‐recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin‐ and flavonol‐acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3‐hydroxyl groups of the acceptor substrates were located at hydrogen‐bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3‐hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5‐ and 7‐hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments.

Generation of blue chrysanthemums by anthocyanin B-ring hydroxylation and glucosylation and its coloration mechanism

Coexpression of two anthocyanin modification genes elicits blue flower coloration through interaction with colorless flavonoids. Various colored cultivars of ornamental flowers have been bred by hybridization and mutation breeding; however, the generation of blue flowers for major cut flower plants, such as roses, chrysanthemums, and carnations, has not been achieved by conventional breeding or genetic engineering. Most blue-hued flowers contain delphinidin-based anthocyanins; therefore, delphinidin-producing carnation, rose, and chrysanthemum flowers have been generated by overexpression of the gene encoding flavonoid 3′,5′-hydroxylase (F3′5′H), the key enzyme for delphinidin biosynthesis. Even so, the flowers are purple/violet rather than blue. To generate true blue flowers, blue pigments, such as polyacylated anthocyanins and metal complexes, must be introduced by metabolic engineering; however, introducing and controlling multiple transgenes in plants are complicated processes. We succeeded in generating blue chrysanthemum flowers by introduction of butterfly pea UDP (uridine diphosphate)–glucose:anthocyanin 3′,5′-O-glucosyltransferase gene, in addition to the expression of the Canterbury bells F3′5′H. Newly synthesized 3′,5′-diglucosylated delphinidin-based anthocyanins exhibited a violet color under the weakly acidic pH conditions of flower petal juice and showed a blue color only through intermolecular association, termed “copigmentation,” with flavone glucosides in planta. Thus, we achieved the development of blue color by a two-step modification of the anthocyanin structure. This simple method is a promising approach to generate blue flowers in various ornamental plants by metabolic engineering.

Functional characterization of UDP-rhamnose-dependent rhamnosyltransferase involved in anthocyanin modification, a key enzyme determining blue coloration in Lobelia erinus.

Summary Because 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′5′‐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. Significance Statement Structural modifications of flavonoids are closely related with their characteristics, but the responsible catalytic enzymes have not been fully investigated in plants. Here we examined enzymatic modifications of a highly glycosylated and acylated anthocyanin, lobelinin, in blue‐flowered Lobelia erinus. We found that multiple modifications of lobelinin were determined by strict substrate specificities of the biosynthetic enzymes and specifically that a rhamnosyltransferase determined a change from blue to mauve.

Anthocyanin and carotenoid pigmentation in flowers of section Mina, subgenus Quamoclit, genus Ipomoea

Plants belong to the section Mina showed characteristic petal pigmentation among Ipomoea plants. For example, petals of Ipomoea hederifolia var. lutea display yellow color derived from carotenoids and those of Ipomoea quamoclit display red color derived from pelargonidin-based anthocyanins. In this study, the pigment composition and the expression patterns of genes for pigment biosynthesis in the petals of I. quamoclit and I. hederifolia var. lutea were analyzed to elucidate the crucial factors that determine petal color in section Mina. The petals of white-flowered I. quamoclit lack the ability to synthesize anthocyanins and carotenoids because of the suppression of anthocyanidin synthase (ANS) gene expression and transcriptional down-regulation of multiple carotenogenic genes, respectively. In petals of I. hederifolia var. lutea, the absence of dihydroflavonol 4-reductase (DFR) gene expression is responsible for the lack of anthocyanins. All F1 progeny obtained by interspecies crossing between I. quamoclit and I. hederifolia var. lutea had scarlet petal color produced by a combination of pelargonidin-based anthocyanins and carotenoids. The flavanone 3-hydroxylase gene, DFR, and ANS were expressed in the petals of the F1 progeny. The results suggest that in these section Mina species, the carotenoid-accumulating phenotype in petals is dominant, and that pelargonidin was produced in the F1 progeny by complementary expression of DFR from white-flowered I. quamoclit and ANS from I. hederifolia var. lutea.

Flower Color and Its Engineering by Genetic Modification

Flower color is mainly determined by the constituent profile of the chemicals flavonoids and the colored subclass of those compounds, the anthocyanins. Flowers often contain specific flavonoids, and thus limited flower colors are available within a species due to genetic constraints. Engineering the flavonoid biosynthetic pathway by expressing a heterologous gene has made it possible to obtain color varieties that cannot be achieved within a species by hybridization or mutational breeding. General tactics for successful engineering flower color have been established on the basis of engineering results obtained in model species such as petunia and torenia. Highly efficient expression of a heterologous gene(s) can be achieved by an optimal combination of promoter, translational enhancer, coding region sequence, and terminator. In addition to expression of heterologous gene, downregulation of competing pathways and/or using color biosynthesis mutant hosts is necessary. As well as a suitable genetic background, it is also important to select hosts with a high market position and value. An efficient transformation system for each target species has to be established. Technical skills and enough finance are also necessary to obtain permits to commercialize genetically modified plants. Violet carnations, roses, and chrysanthemums have been developed by expressing a petunia, pansy, or campanula flavonoid 3′,5′-hydroxylase gene, and genetically modified carnation and rose varieties have been commercialized. Expression of the anthocyanin 3′,5′-glucosyltransferase gene in chrysanthemum in addition to flavonoid 3′,5′-hydroxylase resulted in production of pure blue flower color due to a copigmentation effect with endogenous flavones. Orange petunia expressing maize dihydroflavonol 4-reductase gene and accumulating non-native pelargonidin have been grown worldwide. Though this has been from a non-intentional release of a genetically modified organism, the case provides a good example to show that a combination of genetic engineering and hybridization breeding can produce commercially highly sought after cultivars.