Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1999-03-29
2004-08-10
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C435S320100, C536S023200, C536S023600, C800S282000
Reexamination Certificate
active
06774285
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to genetic sequences encoding flavonoid pathway metabolising enzymes and more particularly to flavonoid 3′-hydroxylase (hereinafter referred to as “F3′H”) or derivatives thereof and their use in the manipulation of pigmentation in plants and other organisms.
Bibliographic details of the publications referred to by the author in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs) for the nucleotide and amino acid sequences referred to in the specification and claims are defined following the bibliography. A summary of the SEQ ID NOs, and the sequences to which they relate, is provided prior to the Examples.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
DESCRIPTION OF THE RELATED ART
The rapidly developing sophistication of recombinant DNA technology is greatly facilitating research and development in a range of biotechnology related industries. The horticultural industry has become a recent beneficiary of this technology which has contributed to developments in disease resistance in plants and flowers exhibiting delayed senescence after cutting. Some attention has also been directed to manipulating flower colour.
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 genetically 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
FIGS. 1
a
and
1
b
(Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979; Wiering and De Vlaming, 1984; Schram et al., 1984; Stafford, 1990; Van Tunen and Mol, 1990; Dooner et al, 1991; Martin and Gerats, 1993; Holton and Cornish, 1995). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA with one molecule of peoumaroyl-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 hydroxylate 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 cytoclrome 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 anthocanins can also result in other flower colours. For example, blue cornflowers contain emcyan. 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 in some instances a single species would be able to produce a broader spectrum of colours.
A nucleotide sequence (referred to herein as SEQ ID NO:26) encoding a petunia flavonoid 3′-hydroxylase has been cloned (see International Patent Application No. PCT/AU93/00127 [WO 93/120206]). However, this sequence was inefficient in its ability to modulate the production of 3′-hydroxylated anhocyanins in plants There is a need, therefore, to identify further genetic sequences encoding flavonoid 3′-hydroxylases which efficiently modulate the hydroxylation of flavonoid compounds in plants. More particularly, there is a need to identify further genetic sequences encoding flavonoid 3′-hydroxylases which efficiently modulate the production of 3′-hydroxylated anthocyanins in plants.
SUMMARY OF THE INVENTION
In accordance with the present invention, genetic sequences encoding flavonoid 3′-hydroxylase have been identified and cloned. The recombinant genetic sequences of the present invention permit the modulation of expression of genes encoding this enzyme by, for example, de novo expression, over-expression, suppression, antisense inhibition and ribozyme activity. The ability to control flavonoid 3′-hydroxylase synthesis in plants permits modulation of the composition of individual anthocyanins as well as alteration of relative levels of flavonols and anthocyanins, thereby enabling the manipulation of tissue colour, such as petals, leaves, seeds and fruit. The present invention is hereinafter described in relation to the manipulation of flower colour but this is done with the understanding that it extends to manipulation of other plant tissues, such as leaves, seeds and fruit.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides encoding a flavonoid 3′-hydroxylase or a derivative thereof wherein said flavonoid 3′-hydroxylase or its derivative is capable of more efficient modulation of hydroxylation of flavonoid compounds in plants than is a flavonoid 3′-hydroxylase encoded by the nucleotide sequence set forth in SEQ ID NO:26.
Efficiency as used herein relates to the capability of the flavonoid 3′-hydroxylase enzyme to hydroxylate flavonoid compounds in a plant cell. This provides the plant with additional substrates for other enzymes of the flavonoid pathway able to further modify this molecule, via, for example, glycosylation, acylation and rhamnosylation, to produce va
Brugliera Filippa
Holton Timothy Albert
Michael Michael Zenon
Florigene Limited
Scully Scott Murphy & Presser
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