Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1994-11-30
1997-06-17
Chereskin, Che S.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
800205, 800DIG67, 800DIG68, C12N 1529
Patent
active
056398700
DESCRIPTION:
BRIEF SUMMARY
The present invention relates generally to genetic sequences encoding flavonoid pathway metabolising enzymes and more particularly to flavonoid 3'-hydroxylase or fragments or derivatives thereof and their use in the manipulation of pigmentation in plants and other organisms.
The flower industry strives to develop new and different varieties of flowering plants. An effective way to create such novel varieties is through the manipulation of flower colour. Classical breeding techniques have been used with some success to produce a wide range of colours for most of the commercial varieties of flowers. This approach has been limited, however, by the constraints of a particular species' gene pool and for this reason it is rare for a single species to have a full spectrum of coloured varieties. In addition, traditional breeding techniques lack precision. The aesthetic appeal of the flower is a combination of many factors such as form, scent and colour; modification of one character through hybridization can often be at the expense of an equally valuable feature. The ability to engineer precise colour changes in cutflower and ornamental species would offer significant commercial opportunities in an industry which has rapid product turnover and where novelty is an important market characteristic.
Flower colour is predominantly due to two types of pigment: flavonoids and carotenoids. Flavonoids contribute to a range of colours from yellow to red to blue. Carotenoids impart an orange or yellow tinge and are commonly the major pigment in yellow or orange flowers. The flavonoid molecules which make the major contribution to flower colour are the anthocyanins which are glycosylated derivatives of cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and are localised in the vacuole. The different anthocyanins can produce marked differences in colour. Flower colour is also influenced by co-pigmentation with colourless flavonoids, metal complexation, glycosylation, acylation and vacuolar pH (Forkmann, 1991).
The biosynthetic pathway for the flavonoid pigments (hereinafter referred to as the "flavonoid pathway") is well established and is shown in FIG. 1 (Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979; Wiering and De Vlaming, 1984; Schram et al., 1984; Stafford, 1990). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA with one molecule of p-coumaroyl-CoA. This reaction is catalysed by the enzyme chalcone synthase (CHS). The product of this reaction, 2',4,4',6', tetrahydroxy-chalcone, is normally rapidly isomerized to produce naringenin by the enzyme chalcone flavanone isomerase (CHI). Naringenin is subsequently hydroxylated at the 3 position of the central ring by flavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK).
The pattern of hydroxylation of the B-ring of DHK plays a key role in determining petal colour. The B-ring can be hydroxylated at either the 3', or both the 3' and 5' positions, to produce dihydroquercetin (DHQ) and dihydromyricetin (DHM), respectively. Two key enzymes involved in this pathway are flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase, both of the cytochrome P450 class. Cytochrome P450 enzymes are widespread in nature and genes have been isolated and sequenced from vertebrates, insects, yeasts, fungi, bacteria and plants.
Flavonoid 3'-hydroxylase acts on DHK to produce DHQ and on naringenin to produce eriodictyol. Reduction and glycosylation of DHQ produces the cyanidin-glycoside and peonidin-glycoside pigments which, in many plant species (for example rose, carnation and chrysanthemum), contribute to red and pink flower colour. The synthesis of these anthocyanins can also result in other flower colours. For example, blue cornflowers contain cyanin. The ability to control flavonoid 3'-hydroxylase activity, or other enzymes involved in the flavonoid pathway, in flowering plants would provide a means to manipulate petal colour. Different coloured versions of a single cultivar could thereby be generated and
REFERENCES:
patent: 5231020 (1993-07-01), Jorgensen et al.
Britsch et al. (1992) "Molecular Cloning, Sequence Analysis and in vitro Expression of Flavanone 3.beta.-Hydroxylase from Petunia hybrida", The Journal of Biological Chemistry 267, 5380-5387.
Bozak, et al. (May 1990) Proc. Natl. Acad. Sci. 87: 3904-3908.
Fugua, et al (1990) Biotechniques 9 (2):206-210.
Cornish Edwina Cecily
Holton Timothy Albert
Tanaka Yoshikazu
Chereskin Che S.
International Flower Developments Pty. Ltd.
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