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

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Science Advances  26 Jul 2017:
Vol. 3, no. 7, e1602785
DOI: 10.1126/sciadv.1602785
  • 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 CamF35H (C) and the resultant delphinidin-based anthocyanins (D). Contents of delphinidin-based anthocyanins corresponded well with the expression of CamF35H. (E and F) Relative expression of CtA35GT (E) and the resultant 3′/3′,5′-glucosylated anthocyanins (F). Contents of 3′/3′,5′-glucosylated anthocyanins corresponded well with the expression of CtA35GT. (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.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/7/e1602785/DC1

    Supplementary Materials and Methods

    fig. S1. Structures of anthocyanins.

    fig. S2. Schematic representation of binary vectors used for chrysanthemum transformation.

    fig. S3. Anthocyanin profiles of representative ‘Sei Arabella’ transgenic lines.

    fig. S4. Flavonoids and caffeoylquinates profiles of representative ‘Sei Arabella’ transgenic lines.

    fig. S5. LC-MS analysis of copigment extract from a cellulose TLC plate.

    fig. S6. Flavonoids and caffeoylquinates profiles of whole and epidermal petal tissue from a blue transgenic line.

    table S1. Flower color changes due to expression of CamF35H and/or CtA35GT.

    table S2. Anthocyanin and flavone content and their ratios in petals of ‘Sei Arabella’ and blue/violet-blue–colored transgenic chrysanthemums.

    table S3. Primer sets used in binary vector construction.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Structures of anthocyanins.
    • fig. S2. Schematic representation of binary vectors used for chrysanthemum transformation.
    • fig. S3. Anthocyanin profiles of representative ‘Sei Arabella’ transgenic lines.
    • fig. S4. Flavonoids and caffeoylquinates profiles of representative ‘Sei Arabella’ transgenic lines.
    • fig. S5. LC-MS analysis of copigment extract from a cellulose TLC plate.
    • fig. S6. Flavonoids and caffeoylquinates profiles of whole and epidermal petal tissue from a blue transgenic line.
    • table S1. Flower color changes due to expression of CamF3′5′H and/or CtA3′5′GT.
    • table S2. Anthocyanin and flavone content and their ratios in petals of ‘Sei Arabella’ and blue/violet-blue–colored transgenic chrysanthemums.
    • table S3. Primer sets used in binary vector construction.

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