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

Abstract

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.

Fig. 1. Scheme for introducing 3′,5′-hydroxylation and 3′,5′-glucosylation into the anthocyanin biosynthetic pathway in chrysanthemum petals.

The major anthocyanin of ‘Taihei’ wild-type chrysanthemum was cyanidin-based anthocyanin (A1). F3'5'H, flavonoid 3',5-hydroxylase; A3′5′GT, UDP-glucose:anthocyanin 3′,5′-O-glucosyltransferase.

Fig. 2. Anthocyanins derived from ray florets of blue-colored transgenic chrysanthemums.

(A) High-performance LC (HPLC) chromatograms of anthocyanins in blue petal extracts detected at 530 nm. (B) Ultraviolet (UV)–visible absorption spectra of A7 and A8 recorded during online HPLC under acidic conditions. An additional spectral maximum at around 320 nm was not detected in either, indicating a lack of aromatic acyl groups. (C) Chemical structures and MS/MS fragmentation spectra of A7 and A8. The product ions of A7 at m/z = 789 [M]⁺ were 627 (−Glc), 465 (−2 × Glc), and 303 (−3 × Glc). The product ions of A8 at m/z = 875 [M]⁺ were 713 (−Glc), 627 (−Glc, −Mal), 465 (−2 × Glc, −Mal), and 303 (−3 × Glc, −Mal; delphinidin aglycone). Dotted arrows show the proposed fragmentation scheme. Glc, glucosyl; Mal, malonyl; A7, delphinidin 3,3′,5′-tri-O-glucoside (preternatin C5); A8, delphinidin 3-O-(6″-O-malonyl)glucoside-3′,5′-di-O-glucoside (ternatin C5).

Fig. 3. Flower color distribution of transgenic ‘Sei Arabella’.

Hue of flower color was evaluated by hue angle (H°), where H° near 270 shows blue color, H° from 300 to 340 shows violet to purple color, and H° over 340 shows red color. Chroma value describes the color strength. The color names given in parentheses are color groups according to the RHS Colour Charts. Circles, wild-type ‘Sei Arabella’; diamonds, transgenic lines.

Fig. 4. Flower coloration, anthocyanin composition, and transgene expression in transgenic ‘Sei Arabella’.

(A) Pictures of representative flower colors of wild-type and transgenic lines. Each line presents the color group and number of RHS Colour Charts. (B) Anthocyanin composition. (C and D) Relative expression of CamF3′5′H (C) and the resultant delphinidin-based anthocyanins (D). Contents of delphinidin-based anthocyanins corresponded well with the expression of CamF3′5′H. (E and F) Relative expression of CtA3′5′GT (E) and the resultant 3′/3′,5′-glucosylated anthocyanins (F). Contents of 3′/3′,5′-glucosylated anthocyanins corresponded well with the expression of CtA3′5′GT. (G) Visible absorption spectra of fresh petals of wild-type (pink), 1916-10 (purple), and 1916-23 (blue) lines. (H) Visible absorption spectra of major anthocyanins derived from pink (A1; wild type), purple-violet (A5), and blue/violet-blue (A8) chrysanthemums in acetate buffer (pH 5.6).

Fig. 5. Copigments responsible for blue coloration associated with anthocyanin.

(A and B) Developed TLC plate observed under fluorescent light (A) and UV light (365 nm) (B). Blue arrows indicate blue color parts on the anthocyanin bands overlapping with band X, which showed yellow fluorescence under UV light. Purple arrows indicate violet color parts overlapping with band Y, which was invisible under fluorescent and UV light. Broken lines indicate regions recovered for LC-MS analyses. (C to E) HPLC chromatograms of extracts from the cellulose TLC plate of band X (C) and band Y (D) and of the blue petal extract (E) monitored by absorption at 360 nm for flavonoids. C1 and C1′ were major compounds detected in band X. C2 and C2′ were major compounds detected in band Y. (F and G) Structures of C1 (F) and C2 (G). Arrows indicate heteronuclear multiple-bond coherence. C1′ and C2′ were identified as demalonyl derivatives of C1 and C2, respectively.

Fig. 6. In vitro reconstruction of petal color by mixing isolated components.

Anthocyanin and copigment were mixed in acetate buffer (pH 5.6). (A) Visible absorption spectra of A8 and its mixture with C1. (B) Visible absorption spectra of A5 and its mixture with C1. (C) Visible absorption spectra of A1 and its mixture with C1. (D) Color of A8 solution (left) and its mixture with C1 (1:10) (right). (E) Color of A5 solution (left) and its mixture with C1 (1:10) (right). (F) Visible absorption spectrum of mixture of A7 or A8 with various flavone glycosides (C1, C2, C1′, and F1′). (G) Visible absorption spectra of a mixture of A8, C1 and C2 solution and its mixture with Fe³⁺ or Mg²⁺ ion. (H) Summary of bathochromic shifts and hyperchromic shifts by A8, A5, and A1 solutions by mixing with C1 (1:5). A8, ternatin C5; A5, delphinidin 3-O-(6′′-O-malonyl)glucoside; A1, cyanidin 3-O-(6″-O-malonyl)glucoside; C1, luteolin 7-O-(6″-O-malonyl)glucoside; C2, tricetin 7-O-(6″-O-malonyl)glucoside; C1′, flavone 7-O-glucoside; F1′, apigenin 7-O-glucoside.