Flower colour and cytochromes P450

Abstract

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

Figure 1.

The flavonoid biosynthetic pathway relevant to flower colour. The pathways leading to anthocyanidin 3-glucosides are conserved in seed plants. Anthocyanidin 3-glucosides are further modified by glycosylation, acylation and methylation in a species-specific manner. P450 enzymes are underlined. In this review, anthocyanins based on peonidin are included as cyanidin-based anthocyanins whilst those based on petunidin and malvidin are included as delphinidin-based anthocyanins. Abbreviations include: PAL, phenylalanine ammonia lyase; C4H, cinnamic acid 4-hydroxylase; CL, 4-coumarate Co-A ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; FNS, flavone synthase; F2H; flavanones 2-hydroxylase; IFS, 2-hydroxyisoflavanone synthase; FLS, flavonol synthase; 3GT, UDP-glucose: anthocyanidin 3-O-glucosyltransferase; MT, S-adenosylmethionine: anthocyanin methyltransferase.

Figure 2.

(a) Phylogenetic tree consisting of CYP75A and CYP75B members from various plant species. F3′5′H and F3′H are shown in blue and red letters, respectively. CYP75A subfamily: Phalaenopsis (CYP75A29, AAZ79451), delphinium (AAX51796), pansy (BAF93855), petunia (CYP75A1, P48418), eggplant (CYP75A2, P37120), butterfly pea (CYP75A24, BAE72870), grape (CYP75A38v4, ABH06585), camellia (AAY23287), cyclamen (ACX37698), periwinkle (CYP75A6, CAA09850), gentian (CYP75A4, BAA12735), lisianthus (CYP75A5, Q96418), torenia (CYP75A10, BAB20076), lavender (CYP75A50, ADA34527), verbena (CYP75A19v2, BAE72871), Kellog's snapdragon (CYP75A48, BAJ16329), canterbury bells (CYP75A6, O04773) and loberia (BAF49321). CYP75B subfamily: carnation (CYP75B28, AAW01411), torenia (CYP75B10, BAB87838), perilla (CYP75B4, BAB59005), snapdragon (CYP75B29, AAW01412), Kellog's snapdragon (CYP75B50, BAB87838), gerbera (CYP75B15, ABA64468), chrysanthemum (AAW01419), China aster F3′H (CYP75B6, AAG49298), cineraria F3′H (CYP75B58), African daisy F3′H (CYP75B14, ABB29899), cineraria F3′5′H (CYP75B18v4), China aster F3′5′H (CYP75B5, AAG49299), African daisy F3′5′H (CYP75B17, ABB43031), camellia (ADZ28515), rose (AAW01418), grape (Q3C211), loberia (BAF49324), morning glory (CYP75B19, BAD00190), gentian (CYP75B9, BAD91808), petunia (CYP75B2, Q9SBQ9), rice (CYP75B3, BAG89180) and Sorghum (CYP75B34, ABG54319). CYP numbers (http://drnelson.uthsc.edu/Nomenclature.html) and/or protein database accession numbers are shown in parenthesis when available. The bar indicates 0.1 substitutions per site. (b) Functional analysis of cDNAs encoding CYP75B18v4 and CYP75B58 derived from blue cineraria petals. Their expression in a petunia line lacking F3′5′H and F3′H activity complemented their deficiency and resulted in colour changes. The two representative flowers expressing the genes (B18-4 and B18-12, B58-9 and B58-23) and the results of their anthocyanidin analysis (c) are shown. Pel, pelargonidin; Cya, sum of cyanidin and peonidin; Del, sum of delphinidin, peonidin and malvidin.

Figure 3.

Commercially launched FLORIGENE Moon series transgenic carnations accumulating delphinidin-based anthocyanins by expressing heterologous F3′5′H genes. The flowers have novel blue/violet colours and accumulate various amounts of delphinidin-derived anthocyanins in their petals resulting in differing petal shades. Binary vector numbers (italic) and commercial names are shown (refer to table 2 for description). Phenotypes of these lines are stable. A flower arrangement consisting of the transgenic carnations is also shown.

Figure 4.

Transgenic rose accumulating delphinidin-based anthocyanins, mainly delphinidin 3,5-diglucoside, by expressing a pansy F3′5′H. (a) Transgenic rose flower expressing the pansy F3′5′H gene (right) derived from a red rose (left). The flower also contained comparable amounts of cyanidin 3,5-diglucoside and had a low vacuolar pH and thus did not show significant blueing. (b) A transgenic rose flower expressing the pansy F3′5′H gene with delphinidin 3,5-diglucosides derived from a pink rose with a higher vacuolar pH than the rose in (a), large amount of flavonols and smaller amount of cyanididin 3,5-diglucoside. The flower has a blue hue that has not been achieved by hybridization breeding [66]. (c) A wedding bouquet containing the novel coloured transgenic rose flowers.

Figure 5.

Transgenic pink torenia flowers accumulating pelargonidin that were generated from torenia accumulating delphinidin-based anthocyanins by (a) suppression of F3′5′H and F3′H genes: (i) blue torenia host, (ii) transgenic flower; and (b) additional expression of pelargonium DFR gene: (i) violet torenia host, (ii) transgenic flower. (c) Field trial of the pink torenia plants. Their growth was comparable with their hosts.