WO2011053167A1

WO2011053167A1 - Transcription factor polynucleotides and their use

Info

Publication number: WO2011053167A1
Application number: PCT/NZ2010/000216
Authority: WO
Inventors: Kerry Ruth Hancock; Vern Eddy Collette
Original assignee: Grasslanz Technology Ltd
Current assignee: Grasslanz Technology Ltd
Priority date: 2009-10-30
Filing date: 2010-10-29
Publication date: 2011-05-05
Anticipated expiration: 2012-04-30
Also published as: UY32995A; AR078857A1

Abstract

The invention provides compositions and methods for manipulating the production of flavonoids, specifically condensed tannins, in plants. The invention provides the isolated polynucleotides encoding the polypeptide sequences of SEQ ID NO: 1 to 5 and variants and fragments thereof. The invention provides the polypeptides encoded by the polynucleotides. The invention also provides, constructs, vectors, host cells, plant cells and plants genetically modified to contain the polynucleotides. The invention also provides methods for producing plants with altered flavonoid, specifically condensed tannin production, making use of polynucleotides of the invention.

Description

TRANSCRIPTION FACTOR POLYNUCLEOTIDES AND THEIR USE

TECHNICAL FIELD

The invention relates to novel compositions and methods for altering the production of flavonoids, particularly condensed tannins, in plants.

BACKGROUND ART

The Molecular Phenylpropanoid Pathway

The phenylpropanoid pathway (shown in Figure 1) produces an array of secondary metabolites including flavones, anthocyanins, flavonoids, condensed tannins and isoflavonoids (Dixon et al., 1996; 2005). In particular, the condensed tannin (CT) biosynthetic pathway shares its early steps with the anthocyanin pathway before diverging to proanthocyanindin biosynthesis.

Condensed Tannins in Forage Plants

Forage plants, such as forage legumes, are beneficial in pasture-based livestock systems because they improve both the intake and quality of the animal diet. Also, their value to the nitrogen (N) economy of pastures and to ruminant production are considerable (Caradus et al., 2000).

However, while producing a cost-effective source of feed for grazing ruminants, pasture is often sub-optimal when it comes to meeting the nutritional requirements of both the rumen microflora and the animal itself. Thus the genetic potential of grazing ruminants for meat, wool or milk production is rarely achieved on a forage diet. White clover {Trifolium repens), red clover {Trifolium pratense) and lucerne (Medicago sativa) are well documented to cause bloat in grazing ruminants, due to the deficiency of plant polyphenolic compounds, such as CTs, in these species. Therefore the development of forage cultivars, particular forage legumes, producing higher levels of tannins in plant tissue would be an important development in the farming industry to reduce the incidence of bloat (Burggraaf et al., 2006).

Condensed tannins, if present in sufficient amounts, not only helps eliminate bloat, but also strongly influences the plant quality, palatability and nutritive value of forage legumes and can therefore help improve animal performance. The animal health and productivity benefits reported from increased levels of CTs include increased ovulation rates in sheep, increased liveweight gain, wool growth and milk production, changed milk composition and improved anthelmintic effects on gastrointestinal parasites (Rumbaugh, 1985; Marten et al., 1987; Niezen et al., 1993; 1995; Tanner et al, 1994; McKenna, 1994; Douglas et al., 1995; Waghorn et al., 1998; Aerts et al,1999; McMahon et al, 2000; Molan et al., 2001 ; Sykes and Coop, 2001).

A higher level of condensed tannin also represents a viable solution to reducing greenhouse gases (methane, nitrous oxide) released into the environment by grazing ruminants (Kingston- Smith and Thomas, 2003). Ruminant livestock produce at least 88% of New Zealand's total methane emissions and are a major contributor of greenhouse gas emissions (Clark, 2001). The principle source of livestock methane is enteric fermentation in the digestive tract of ruminants. Methane production, which represents an energy loss to ruminants of around 3 to 9% of gross energy intake (Blaxter and Clapperton, 1965), can be reduced by as much as 5% by improving forage quality. Forage high in CT has been shown to reduce methane emission from grazing animals (Woodward, et al 2001 ; Puchala, et al., 2005). Increasing the CT content of pasture plants can therefore contribute directly to reduced levels of methane emission from livestock.

Therefore, the environmental and agronomical benefits that could be derived from triggering the accumulation of even a moderate amount of condensed tannins in forage plants are of

considerable importance in the protection and nutrition of ruminants (Damiani et al., 1999).

Legumes The legume genus, Medicago, is one genera in the family Leguminosae (D Fabaceae), with ca. 58 recognised species (Small, E. & M. Jomphe. 1989). Medicago sativa (commonly known as Alfalfa or lucerne) is the most cultivated legume in the world. Worldwide production was around 454 million tons in 2002 (FAO). The US is the largest alfalfa producer in the world, but considerable area is found in Argentina (primarily grazed), Australia, South Africa, and the Middle East. Alfalfa, called the "Queen of the Forages," is the fourth most widely grown crop in the United States behind corn, wheat and soybeans and double the cotton acreage.

It would be of considerable benefit to be able to manipulate production of flavonoids, particularly condensed tannins in plants, particularly forage plants, and particularly legumes including Medicago species. It is an object of the invention to provide improved compositions and methods for altering the production of flavonoids, particularly condensed tannins in plants, particularly legumes such as Medicago species, or at least to provide the public with a useful choice. SUMMARY OF THE INVENTION

The present invention is concerned with the identification and uses of a novel MYB transcription factor gene and associated polypeptide which has-faeen isolated and identified by the applicants as being useful for manipulating production of flavonoid compounds, including condensed tannins, in plants.

Polynucleotides encoding polypeptides

In a first aspect the invention provides an isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 1 to 5 or a variant or fragment thereof.

In one embodiment polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO: 1 or a variant or fragment thereof.

In a further embodiment polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO:2 or a variant or fragment thereof. In a further embodiment polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO: 3 or a variant or fragment thereof.

In a further embodiment polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO:4 or a variant or fragment thereof.

In a further embodiment polynucleotide encodes a polypeptide with the amino acid sequence of SEQ ID NO: 5 or a variant or fragment thereof.

Preferably the polypeptide is a MYB transcription factor.

Preferably the MYB transcription. factor is an R2R3 MYB transcription factor.

Preferably the R2R3 MYB transcription factor contains the amino acid pair TQ (threonine glutamate) in the R2 repeat of the R2R3 DNA binding domain.

Preferably T and Q are at amino acid positions 20 and 21 respectively in the R2R3 MYB transcription factor polypeptide. Preferably the polypeptide is capable of positively regulating production of at least one flavonoid in a plant. In a preferred embodiment the flavonoid is a condensed tannin.

In one embodiment the variant has at least 70% identity to the amino acid sequence of any one of SEQ ID NO: 1 to 5. In a further embodiment the variant has at.least 70% identity to the amino acid sequence of SEQ II) NO: 1 .

In a further embodiment the variant has at least 70% identity to the amino acid sequence of SEQ ID NO: 2.

In a further embodiment the variant has at least 70% identity to the amino acid sequence of SEQ ID NO: 3.

In a further embodiment the variant has at least 70% identity to the amino acid sequence of SEQ ID NO: 4.

In a further embodiment the variant has at least 70% identity to the amino acid sequence of SEQ ID NO: 5.

In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO:22

In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO:23 In a further embodiment the variant comprises the amino acid sequence of both SEQ ID NO:22 and SEQ ID NO:23

In a further embodiment the variant is derived from a Medicago species. In a further embodiment the variant of SEQ ID NO:l comprises the amino acid sequence of any oneofSEQIDNO:2to5.

In a further embodiment the variant of SEQ ID NO:2 comprises the amino acid sequence of any one of SEQ ID NO: 1 and 3 to 5.

In a further embodiment the variant of SEQ ID NO:2 comprises the amino acid sequence of any oneofSEQIDNO:3and4. In a further embodiment the variant of SEQ ID NO:3 comprises the amino acid sequence of any one of SEQ ID NO: 1, 2, 4 and 5.

In a further embodiment the variant of SEQ ID NO:3 comprises the amino acid sequence of any oneofSEQIDNO:2and4.

In a further embodiment the variant of SEQ ID NO:4 comprises the amino acid sequence of any one of SEQ ID NO: 1 to 3 and 5.

In a further embodiment the variant of SEQ ID NO:4 comprises the amino acid sequence of any oneofSEQIDNO:2and3.

In a further embodiment the variant of SEQ ID NO:5 comprises the amino acid sequence of any one of SEQ ID NO: 1 to 4. In a preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 5.

Polynucleotides

In a further aspect the invention provides an isolated polynucleotide comprising the sequence of any one of SEQ ID NO: 6 to 15 or a variant or fragment thereof.

Preferably the polynucleotide encodes a MYB transcription factor.

Preferably the polynucleotide encodes a R2R3 MYB transcription factor.

-

Preferably T and Q are at amino acid positions 20 and 21 respectively in the R2R3 MYB transcription factor polypeptide.

Preferably the polynucleotide encodes a polypeptide that is capable of positively regulating production of at least one flavonoid in a plant. In a preferred embodiment the flavonoid is a condensed tannin.

In one embodiment the variant has at least 70% identity to the sequence of any one of SEQ ID NO: 6 to 15. In a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 6. In a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 8.

In a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 10. .

In a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 12. In a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 14.

In a further embodiment the variant is derived from a Medicago species.

In a further embodiment the variant of SEQ ID NO:6 comprises the sequence of any one of SEQ ID NO: 8, 10, 12 and 14.

In a further embodiment the variant of SEQ ID NO:8 comprises the sequence of any one of SEQ ID NO: 6, 10, 12 and 14.

In a further embodiment the variant of SEQ ID NO:8 comprises the sequence of any one of SEQ ID NO: 10 to 13.

In a further embodiment the variant of SEQ ID NO: 10 comprises the sequence of any one of SEQ ID NO: 6, 8, 12 and 14.

In a further embodiment the variant of SEQ ID NO: 10 comprises the sequence of any one of SEQ ID NO: 8, 9, 12 and 13.

In a further embodiment the variant of SEQ ID NO: 12 comprises the sequence of any one of SEQ ID NO: 6, 8, 10, and 14.

In a further embodiment the variant of SEQ ID NO: 12 comprises the sequence of any one of SEQ ID NO: 8 to 1 1. In a further embodiment the variant of SEQ ID NO: 14 comprises the sequence of any one of SEQ ID NO: 6, 8, 10, and 12.

In a preferred embodiment the polynucleotide comprises the sequence of SEQ ID NO: 6. In an alternative preferred embodiment the polynucleotide comprises the sequence of SEQ ID NO: 8.

In an alternative preferred embodiment the polynucleotide comprises the sequence of SEQ ID NO: 10.

In an alternative preferred embodiment the polynucleotide comprises the sequence of SEQ ID

NO: 12. ^■■■_¾ ^{■ ■} In an alternative preferred embodiment the polynucleotide comprises the sequence of SEQ ID NO: 14.

Polypeptides

In a further aspect the invention provides an isolated polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 1 to 5 or a.variant or fragment thereof.

In one embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or a variant or fragment thereof. In one embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or a variant or fragment thereof.

In one embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or a variant or fragment thereof.

In one embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or a variant or fragment thereof. ,

In one embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 5 or a variant or fragment thereof.

Preferably the polypeptide or variant is a MYB transcription factor.

Preferably the MYB transcription factor is an R2R3 MYB transcription factor. Preferably the R2R3 MYB transcription factor contains the amino acid pair TQ (threonine glutamate) in the R2 repeat of the R2R3 DNA binding domain. Preferably T and Q are at amino acid positions 20 and 21 respectively in the R2R3 MYB transcription factor polypeptide.

Preferably the polypeptide is capable of positively regulating production of at least one flavonoid in a plant. In a preferred embodiment the flavonoid is a condensed tannin.

In one embodiment the variant has at least 70% identity to the amino acid sequence of any one of SEQ ID NO: 1 to 5.

-

In a further embodiment the variant has at least 70% identity to. the amino acid sequence of SEQ ID NO: 1. ^"

In a further embodiment the variant has at least 70% identity to the amino acid sequence of SEQ ID NO: 3. In a further embodiment the variant has at least 70% identity to the amino acid sequence of SEQ - ID NO: 4.

In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO:22 In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO:23 In a further embodiment the variant comprises the amino acid sequence of both SEQ ID NO:22 and SEQ ID NO:23

In a further embodiment the variant is derived from a Medicago species.

In a further embodiment the variant of SEQ ID NO:l comprises the amino acid sequence of any one of SEQ ID NO: 2 to 5.

In a preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 4.

In a further aspect the invention provides an antibody raised against a polypeptide of the invention.

In a further aspect the invention provides an isolated polynucleotide encoding a polypeptide of the invention.

In a further aspect the invention provides an isolated polynucleotide comprising:

a) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of a polynucleotide of the invention;

b) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of the invention; or d) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of the invention.

In a further aspect the invention provides a genetic construct comprising a polynucleotide of the invention.

In a further aspect the invention provides an expression construct comprising a polynucleotide of the invention.

In a further aspect the invention provides an RNAi construct comprising a polynucleotide of the invention.

In a further aspect the invention provides a vector comprising an expression construct, genetic construct or RNAi construct of the invention.

In a further aspect the invention provides a host cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention.

In a further aspect the invention provides a host cell comprising an expression construct, RNAi construct, or genetic construct of the invention.

Preferably the host cell does not form part of a human being.

In one embodiment the host cell is a plant cell.

In a further aspect the invention provides a plant comprising a plant cell of the invention.

In a further aspect the invention provides a plant genetically modified to express a polynucleotide of the invention, or polypeptide of the invention.

In a further aspect the invention provides a plant comprising an expression construct, RNAi construct, or genetic construct of the invention. In a further aspect the invention provides a part, propagule, seed, fruit, progeny, or harvested material derived from the plant of the invention.

In one embodiment the plant part, propagule, seed, fruit, progeny, or harvested material is genetically modified to comprise at least one polynucleotide, polypeptide, expression construct, - RNAi construct, or genetic construct of the invention.

Methods - polynucleotides encoding polypeptides In a further aspect the invention provides a method for producing a plant cell or plant with altered flavonoid content, the method comprising transformation of a plant cell, or plant, with a polynucleotide encoding a polypeptide with the amino acid sequence of any one of SEQ ID NO: 1 to 5, or a variant or fragment thereof. Preferably the polypeptide is a MYB transcription factor _ ^■ .

Preferably the MYB transcription factor is an R2R3 MYB transcription factor.

Preferably T and Q are at amino acid positions 20 and 21 respectively in the R2R3 MYB transcription factor polypeptide. Preferably the polypeptide is capable of positively regulating production of at least one flavonoid in a plant.

In a preferred embodiment the flavonoid is a condensed tannin.

In one embodiment the variant has at least 70% identity to the amino acid sequence of any one of ^~- SEQ ID NO: 1 to 5.

In a further embodiment the variant has at least 70% identity to the amino acid sequence of SEQ ID NO: l . In a further embodiment the variant has at least 70% identity to the amino acid sequence of SEQ ID NO: 2. In a further embodiment the variant has at least 70% identity to the amino acid sequence of SEQ ID NO: 3.

- _. ^■

In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO:23

In a further embodiment the variant comprises the amino acid sequence of both SEQ ID NO:22 and SEQ ID NO:23

■

In a preferred embodiment the variant is derived from a Medicago species.

In a further embodiment the variant of SEQ ID NO: l comprises the amino acid sequence of any one of SEQ ID NO: 2 to 5.

In a further embodiment the variant of SEQ ID NO:2 comprises the amino acid sequence of any one of SEQ ID NO: 1 and 3 to 5^

In a further embodiment the variant of SEQ ID NO;2 comprises the amino acid sequence of any one of SEQ ID NO: 3 and 4.

In a further embodiment the variant of SEQ ID NO: 3 comprises the amino acid sequence of any one of SEQ ID NO: 1, 2, 4 and 5. In a further embodiment the variant of SEQ ID NO:3 comprises the amino acid sequence of any one of SEQ ID NO: 2 and 4.

In a further embodiment the variant of SEQ ID NO:4 comprises the amino acid sequence of any one of SEQ ID NO: 2 and 3. In a further embodiment the variant of SEQ ID NO:5 comprises the amino acid sequence of any one of SEQ ID NO: 1 to 4.

In a preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 .

In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 3.

In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In an alternative preferred embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO: 5.

In a preferred embodiment the flavonoid content is increased in the plant produced. In a further embodiment flavonoid is produced in a foliar tissue.

In a further embodiment flavonoid is produced in a leaf.

In a further embodiment flavonoid is produced in an epidermal tissue. In a further embodiment the flavonoid is produced in a tissue, or cell type, that is substantially devoid of the flavonoid, in a control plant, such as a similar plant that has not been transformed. Methods - polynucleotides

In a further aspect the invention provides a method for producing a plant cell or plant with altered flavonoid content, the method comprising transformation of a plant cell or plant with an isolated polynucleotide comprising the sequence of any one of SEQ ID NO: 6 to 15 or a variant or fragment thereof.

Preferably the polynucleotide encodes a MYB transcription factor.

Preferably the polynucleotide encodes a R2R3 MYB transcription factor.

Preferably the polynucleotide encodes a polypeptide that is capable of positively regulating production of at least one flavonoid in a plant. In a preferred embodiment the flavonoid is condensed tannin.

In one embodiment the variant has at least 70% identity to the sequence of any one of SEQ ID NO: 6 to 15. In a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 6. In a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 8. a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 10. In a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 12. In a further embodiment the variant has at least 70% identity to the sequence of SEQ ID NO: 14.

In a preferred embodiment the variant is derived from a Medicago species.

In a further embodiment the variant of SEQ ID NO:6 comprises the sequence of any one of SEQ ID NO: 8 to 15.

In a further embodiment the variant of SEQ ID NO: 10 comprises the sequence of any one of SEQ ID NO: 6, 8, 12 and 14. In a further embodiment the variant of SEQ ID NO: 10 comprises the sequence of any one of SEQ ID NO: 8. 9, 12 and 13.

In a further embodiment the variant of SEQ ID NO: 12 comprises the sequence of any one of SEQ ID NO: 8 to 11.

In a further embodiment the variant of SEQ ID NO: 14 comprises the sequence of any one of SEQ ID NO: 6 to 13.

a preferred embodiment the polynucleotide comprises the sequence of SEQ ID NO: 6. In an alternative preferred embodiment the polynucleotide comprises the sequence of SEQ ID NO: 8. In an alternative preferred embodiment the polynucleotide comprises the sequence of SEQ ID NO: 10.

In an alternative preferred embodiment the polynucleotide comprises the sequence of SEQ ID NO: 12.

In an alternative preferred embodiment the polynucleotide comprises the sequence of SEQ ID NO: 14.

In a preferred embodiment the flavonoid content is increased in the plant produced.

In a further embodiment flavonoid is produced in a foliar tissue.

In a further embodiment flavonoid is produced in a leaf. In a further embodiment flavonoid is produced in an epidermal tissue.

In a further embodiment the flavonoid is produced in a tissue, or cell type, that is substantially devoid of the flavonoid, in a control plant, such as a similar plant that has not been transformed.

Method for. producing a flavonoid in a host cell In a further aspect the invention provides a method for producing a flavonoid, the method comprising culturing a host cell comprising an expression construct of the invention or a genetic construct of the invention under conditions in which a polynucleotide or polypeptide of the invention is expressed. Preferably the host cell is a plant cell. Preferably the plant cell is part of a plant.

It may be desirable, in some cases, to reduce flavonoid content in a plant. This can be achieved by down-regulating expression of endogenous gene or nucleic acid comprising a polynuleotide of the invention, or encoding a polypeptide of the invention. Methods for down-regulation or silencing endogenous genes in plants are well known to those skilled in the art and described herein. Methods for reducing flavonoid content in plant cells or plants, are also included within the scope of the invention.

Preferably the flavonoid is a condensed tannin.

Plants produced via the methods ,

In a further embodiment the invention provides a plant produced by a method of the invention.

In a further embodiment the invention provides a part, seed, fruit, harvested material, propagule or progeny of a plant of any the invention.

In a further embodiment the part, seed, fruit, harvested material, propagule or progeny of the plant is genetically modified to comprise at least one polynucleotide of the invention, or construct of the invention.

Selection methods

In a further aspect the invention provides a method for selecting a plant with altered flavonoid content, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.

In a further aspect the invention provides a method for selecting a plant with altered flavonoid content, the method comprising testing of a plant for altered expression of a polypeptide of the invention.

In a further aspect the invention provides a group or population of plants selected by the method of the invention.

Source of polynucleotides and polypeptides of the invention

The polynucleotides and polypeptides of the invention may be derived from any plant, as described below, or may be synthetically or recombinantly produced.

Plants The plant cells and plants of the invention, or those transformed or manipulated in methods and uses of the inventions, may be from any species.

In one embodiment the plant cell or plant, is derived from a gymnosperm plant species.

In a further embodiment the plant cell or plant, is derived from an angiosperm plant species.

In a further embodiment the plant cell or plant, is derived from a from dicotyledonous plant species.

·

In a further embodiment the plant cell or plant, is derived from a monocotyledonous plant species.

Preferably the plants are from dicotyledonous species. Other preferred plants are forage plant species from a group comprising but not limited to the following genera: Lolium, Festuca, Dactylis, Bromus, Thinopyrum, Trifolium, Medicago, Pheleum, Phalaris, Holcus, Lotus, Plantago and Cichorium.

Other preferred plants are leguminous plants. The leguminous plant or part thereof may encompass any plant in the plant family Leguminosae or Fabaceae. For example, the plants may be selected from forage legumes including, alfalfa, clover; leucaena; grain legumes including, beans, lentils, lupins, peas, peanuts, soy bean; bloom legumes including lupin, pharmaceutical or industrial legumes; and fallow or green manure legume species.

A preferred genus is Trifolium. Preferred Trifolium species include Trifolium repens; Trifolium arvense; Trifolium affine; and Trifolium occidentale. A preferred Trifolium species is Trifolium repens.

Another preferred genus is Glycine. Preferred Glycine species include Glycine max and Glycine wightii (also known as Neonotonia wightii). A particularly preferred Glycine species is-Glycine wightii, commonly known as perennial soybean. Another preferred genus is Vigna. A particularly preferred Vigna species is Vigna unguiculata, commonly known as cowpea.

Another preferred genus is Mucana. A preferred Mucana species is Mucana pruniens commonly known as velvetbean. Another preferred genus is Arachis. A preferred Arachis species is Arachis glabrata commonly known as perennial peanut.

Another preferred genus is Pisum. A particularly preferred Pisum species is Pisum sativum, commonly known as pea.

Another preferred genus is Lotus. Preferred Lotus species include Lotus corniculatus, Lotus pedunculatus, Lotus glabar, Lotus tenuis and Lotus uliginosus. A particularly preferred Lotus species is Lotus corniculatus, commonly known as Birdsfoot Trefoil. Another preferred Lotus species is Lotus glabar, commonly known as Narrow-leaf Birdsfoot Trefoil. Another preferred Lotus species is Lotus pedunculatus, commonly known as Big trefoil. Another preferred Lotus species is Lotus tenuis, commonly known as Slender trefoil.

Another preferred genus is Brassica. A particularly preferred Brassica species is Brassica oleracea, commonly known as forage kale and cabbage.

A particularly preferred genus is Medicago. _, Preferred Medicago species include Medicago sativa and Medicago truncatula. A particularly preferred Medicago species is Medicago sativa, commonly known as alfalfa, or lucerne.

DETAILED DESCRIPTION

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art. The term "comprising" as used in this specification and claims means "consisting at least in part of; that is to say when interpreting statements in this specification and claims which include "comprising", the features prefaced by this term in each statement all need to be present but other features can also be present. Related terms such as "comprise" and "comprised" are to be interpreted in similar manner. However, in preferred embodiments comprising can be replaced with consisting.

The term "MYB transcription factor" is a term well understood by those skilled in the art to refer to a class of transcription factors characterised by a structurally conserved DNA binding domain consisting of single or multiple imperfect repeats. MYB proteins can be classified into three subfamilies depending on the number of adjacent repeats in the MYB domain.

The term "R2R3 transcription factor" or "MYB transcription with an R2R3 DNA binding domain" is a term well understood by those skilled in the art to refer to MYB transcription factors of the two-repeat class (Stracke et al., 2001). There are 133 R2R3 MYB TF-encoding genes in Arabidopsis classified into 24 subgroups.

The term "flavonoids" as used herein includes but is not restricted to known flavanols such as catechin, epicatechin, epigallocatechin and gallocatechin derived from procyanidin ^" and prodelphinidinand, and their polymeric forms known as condensed tannins. Preferably the flavonoid is a condensed tannin.

The terms 'condensed tannins' and 'proanthocyanidins' may be used interchangeably throughout the specification. The term "condensed tannin" includes: any form of oligomeric and polymeric flavanol formed by condensation of monomeric units such as flavan-3-ols and flavan-3-4-diols. This includes but is not restricted to known flavanols such as catechin, epicatechin, epigallocatechin and gallocatechin derived from procyanidin and prodelphinidin

The term "sequence motif as used herein means a stretch of amino acids or nucleotides.

Preferably the stretch of amino acids or nucleotides is contiguous. The term "altered" with respect to a plant with "altered production" or "altered expression", means altered relative to the same plant, or plant of the same type, in the non-transformed state.

The term "altered" may mean increased or decreased. Preferably altered is. increased

The phrase "capable of positively regulating flavonoid production or content or grammatical equivalents thereof with respect to a polypeptide or polynucleotide, means that when the polypeptide or polynucleotide is expressed in a cell, plant cell or plant, there is an increase in the production, biosynthesis or content of a flavonoid in the cell, plant cell or plant. Typically over- expression of polynucleotide or polypeptide via genetic manipulation will result in increased flavonoid production, biosynthesis or content.

The term "control plant" includes a non-transformed plant of the same type, genus, species or variety. The control plant may be a control plant transformed with a control construct, such as an empty vector construct.

The term "epidermal tissue" refers to the outer single-layered group of cells, including the leaf, stems, and roots and young tissues of a vascular plant. Polynucleotides and fragments _. ^' _.

The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments. The term "polynucleotide" can be used interchangably with "nucleic acid molecule".

A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is preferably at least 15 nucleotides in length. The fragments of the invention preferably comprises at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 contiguous nucleotides of . a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods.

Preferably fragments of polynucleotide sequences of the invention comprise at least 25, more preferably at least 50, more preferably at least 75, more preferably at least 100, more preferably at least 150, more preferably at least 200, more preferably at least 300, more preferably at least 400, more preferably at least 500, more preferably at least 600, more preferably at least 700, more preferably at least 800, more preferably at least 900, more preferably at least 1000 contiguous nucleotides of the specified polynucleotide.

The term "primer" refers to a short polynucleotide, usually having a free 3ΌΗ group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the template. Such a primer is preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 1 1, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length.

The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence, that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein. Preferably such a probe is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and most preferably at least 500 nucleotides in length.

'

Polypeptides and fragments -

The term "polypeptide", as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. The polypeptides may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.

· ·

A "fragment" of a polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof capable of performing the above activity.

The term "isolated" as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.

The term "derived from" with respect to a polynucleotide or polypeptide sequence being derived from a particular genera or species, means that the sequence has the same sequence as a polynucleotide or polypeptide sequence found naturally in that genera or species. The sequence, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.

Variants As used herein, the term "variant" refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the inventive polynucleotides and polypeptides possess biological activities that are the same or similar to those of the inventive polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein. Polynucleotide variants

Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least_. 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, more preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, more preferably at least 200 nucleotide positions, more preferably at least 300 nucleotide positions, more preferably at least 400 nucleotide positions, more preferably at least 500 nucleotide positions, more preferably at least 600 nucleotide positions, more preferably at least 700 nucleotide positions, more preferably at least 800 nucleotide positions, more preferably at least 900 nucleotide positions, more preferably at least 1000 nucleotide positions and most preferably over the entire length of the specified polynucleotide sequence. Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.

The identity of polynucleotide sequences may be examined using the following unix command line parameters: bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn

The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in- a line "Identities = ".

Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment · programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman- Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. LongdenJ. and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276- 277) which can be obtained from http://www.hgmp.mrc.ac.uk Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle^' global alignments between two sequences on line at http:/www.ebi. ac.uk/emboss/align/.

Alternatively the GAP program, which computes an optimal global alignment of two sequences without penalizing terminal gaps, may be used to calculate sequence identity. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.

Sequence identity may also be calculated by aligning sequences to be compared using Vector NTI version 9.0, which uses a Clustal W algorithm (Thompson et al., 1994, Nucleic Acids Research 24, 4876-4882), then calculating the percentage sequence identity between the aligned sequences using Vector NTI version 9.0 (Sept 02, 2003 ©1994-2003 InforMax, licenced to Invitrogen).

Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polynucleotides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 20021) from NCBI (ftp://ftp.ncbi.nih.gov/blast/).

The similarity of polynucleotide sequences may be examined using the following unix command line parameters: bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p tblastx

The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.

Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10 ^"10 more preferably less than 1 x 10 ^"20, more preferably less than 1 10 ^"30, more preferably less than 1 x 10 ^"40, more preferably less than 1 x 10 ^"50 more preferably less than 1 x 10 ^"60 more preferably less than 1 x 10 ^"70 more preferably less than 1 x 10 ^~80 more preferably less than 1 x 10 ^"90 and most preferably less than 1 x 10 ^"10° when compared with any one of the specifically identified sequences.

Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions. The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.

With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C (for example, 10° C) below the melting temperature (Tm) of the native duplex (see generally, S.ambrook et al, Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al, 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al, Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84: 1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65° C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.

With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)⁰ C.

With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al, Science. 1991 Dec 6;254(5037): 1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov l ;26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10° C below the Tm.

Variant polynucleotides such as those in constructs of the invention encoding proteins to be expressed, also encompasses polynucleotides that differ from the specified sequences but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also contemplated. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al, 1990, Science 247, 1306).

Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI ( ftp://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.

Polypeptide variants

The . term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences^' preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least .65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, . more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%), and most preferably at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length of a polypeptide of the invention.

Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www.ebi. ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227- 235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

. . ^{" ■} . ·

Sequence identity may also be calculated by aligning sequences to be compared using Vector NTI version 9.0, which uses a Clustal W algorithm (Thompson et al., 1994, Nucleic Acids Research 24, 4876-4882), then calculating the percentage sequence identity between the aligned polypeptide sequences using Vector NTI version 9.0 (Sept 02, 2003 ©1994-2003 InforMax, licenced to Invitrogen).

Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the. publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences may be examined using the following unix command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 ^"6 more preferably less than 1 x 10 ^" , more preferably less than 1 x 10 ^" , more preferably less than 1 x 10 ^"l5, more preferably less than 1 x 10 ^"18, more preferably less than 1 x 10 ^"21, more preferably less than 1 x 10 ^"30, more preferably less than 1 x 10 ^⁰, more preferably less than 1 x 10 ^"50, more preferably less than 1 x 10 ^"60, more preferably less than 1 x 10 ^"70, more preferably less than 1 x 10 ^"80, more preferably less than 1 x 10 ^:90 and most preferably 1x10^{" 100} when compared with any one of the specifically identified sequences.

The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al. , 1990, Science 247, 1306). The function of polynucleotides, polypeptides and variants or fragments thereof in manipulating production of flavonoids, such as condensed tannins, in plants can be tested by methods known to those skilled in the art, and described in the Examples section of this specification.

Constructs, vectors and components thereof

The term "genetic construct" refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain a promoter polynucleotide including the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a synthetic or recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.

The term "vector" refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system, such as E. coli.

The term "expression construct" refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide.

An expression construct typically comprises in a 5' to 3' direction:

a) a promoter functional in the host cell into which the construct will be transformed,

b) the ρο^ημοΐεο^ε to be expressed, and

c) a terminator functional in the host cell into which the construct will be transformed.

The term "coding region" or "open reading frame" (O F) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a genetic construct, a "coding sequence" is capable of being expressed when it is operably linked to promoter and terminator sequences. The term "operably-linked" means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators. The term "noncoding region" includes to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5' UTR and the 3' UTR. These sequences may include elements required for transcription initiation and termination and for regulation of translation efficiency. The term "noncoding" also includes intronic sequences within genomic clones.

-

Terminators are sequences, which terminate transcription, and are found in the 3' untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions. The term "promoter" refers to a polynucleotide sequence capable of regulating or driving the expression of a polynucleotide sequence to which the promoter is operably linked in a cell, or cell free transcription system. Promoters may comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors. Methods for isolating or producing polynucleotides -

The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polynucleotides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al, Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polynucleotides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.

Further methods for isolating polynucleotides of the invention, or useful in the methods of the invention, include use of all or portions, of the polynucleotides set forth herein as hybridization probes. The technique of hybridizing labeled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65°C in 5. 0 X SSC, 0. 5% sodium dodecyl sulfate, 1 X Denhardt's solution; washing (three washes of twenty minutes each at 55°C) in 1. 0 X SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0. 5 X SSC, 1 % (w/v) sodium dodecyl sulfate, at 60°C. An optional further wash (for twenty minutes) can be conducted under conditions of 0. 1 X SSC, 1% (w/v) sodium dodecyl sulfate, at 60°C. The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion, oligonucleotide synthesis and PCR amplification.

A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full length polynucleotide sequence and/or the whole gene/ and/or the promoter. Such methods include PCR-based methods, 5'RACE (Frohman MA, 1993, Methods Enzymol. 218: 340-56) and hybridization- based method, computer/database -based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et ah, 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a polynucleotide. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. Promoter and flanking sequences may also be isolated by PCR genome walking using a GenomeWalker™ kit (Clontech, Mountain View, California), following the manufacturers instructions. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).

It may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms. Additionally when down-regulation of a gene is the desired result, it may be necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species. Variants (including orthologues) may be identified by the methods described.

Methods for identifying variants Physical methods

Variant polynucleotides may be identified using PCR-based methods (Mullis et al , Eds. 1994 The Polymerase Chain Reaction, Birkhauser).

Alternatively library screening methods, well known to those skilled in the art, may be employed (Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). When identifying variants of the probe sequence, hybridization and/or wash stringency will typically be reduced relatively to when exact sequence matches are sought.

Computer-based methods

Polynucleotide and polypeptide variants may also be identified by computer-based methods well-known to those skilled in the_ art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1 -10 and 1 1-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.

An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih.gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894 USA. The NCBI server also provides the facility to use .the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six- frame translations of a nucleotide query sequence against the six-frame: translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen. The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al, Nucleic Acids Res. 25: 3389-3402, 1997. The "hits" to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.

The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce "Expect" values for alignments. The Expect value (E) indicates the number of hits one can "expect" to see by chance when searching a database of the same size containing random contiguous sequences. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide . hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.

Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673- 4680, http://www-igbmc.u-strasbg.fr/BioInfo/ClustalW/Top.html) or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217)) or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).

Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found. MEME and MAST were developed at the University of California, San Diego.

PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al, 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (www.expasy.org/prosite) contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.

Methods for producing constructs and vectors

The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynucleotides encoding polypeptides disclosed, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or particularly plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined. Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing, 1987). Methods for producing host cells comprising constructs and vectors

The invention provides a host cell which comprises a genetic construct or vector of the invention. Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.

Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al, Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al, Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutseher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).

Methods for producing plant cells and plants comprising constructs and vectors

The invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide. Plants comprising such cells also form an aspect of the invention.

Methods for transforming plant cells, plants and portions thereof with polynucleotides are described in Draper et al, 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer- Verlag, Berlin.; and Gelvin et al, 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.

The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); apple (Yao et al, 1995, Plant Cell Reports 14, 407-412); maize (US Patent Serial Nos. 5, 177, 010 and 5, 981, 840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (US Patent Serial No. 5, 1.59, 135); potato (Kumar et al., 1996 Plant J. 9, : 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al, 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al, 1985, Science 227, 1229); cotton (US Patent Serial Nos. 5, 846, 797 and 5, 004, 863); perennial ryegrass (Bajaj et al , 2006, Plant Cell Rep. 25, 651); grasses (US Patent Nos. 5, 187, 073, 6. 020, 539); peppermint (Niu et al, 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al, 1995, Plant Sci.104, 183); caraway (Krens et al, 1997, Plant Cell Rep, 17, 39); banana (US Patent Serial No. 5, 792, 935); soybean (US Patent Nos. 5, 416, 01 1 ; 5, 569, 834 ; 5, 824, 877 ; 5, 563, 04455 and 5, 968, 830); pineapple (US Patent Serial No. 5, 952, 543); poplar (US Patent No. 4, 795, 855); monocots in general (US Patent Nos. 5, 591, 616 and 6, 037, 522); brassica (US Patent Nos. 5, 188, 958 ; 5, 463, 174 and 5, 750, 871); and cereals (US Patent No. 6, 074, 877); pear (Matsuda et al, 2005, Plant Cell Rep. 24(1):45-51); Prunus (Ramesh et al., 2006, Plant Cell Rep. 25(8):821-8; Song and Sink 2005, Plant Cell Rep. 2006; 25(2): 1 17-23; Gonzalez Padilla et al, 2003, Plant Cell Rep. 22(l):38-45); strawberry (Oosumi et al, 2006, Planta.; 223(6): 1219-30; Folta et al., 2006, Planta. 2006 Apr 14; PMID: 16614818), rose (Li et al, 2003, Planta. 218(2):226-32), Rubus (Graham et al, 1995, Methods Mol Biol. 1995;44: 129-33). Clover (Voisey et al, 1994, Plant Cell Reports 13 : 309-314, and Medicago (Bingham, 1991 , Crop Science 31 : 1098). Transformation of other species is also contemplated by the invention. Suitable methods and protocols for transformation of other species are available in the scientific literature.

Methods for genetic manipulation of plants A number of strategies for genetically manipulating plants are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297). For example, strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed. Strategies may also be designed to increase expression of a polynucleotide/polypeptide in response to external stimuli, such as environmental stimuli. Environmental stimuli may include environmental stresses such as mechanical (such as herbivore activity), dehydration, salinity and temperature stresses. The expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.

Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed or to reduce expression of a polynucleotide/polypeptide in response to an external stimuli. Such strategies are known as gene silencing strategies.

Genetic constructs for expression of genes in transgenic plants typically include promoters, such as promoter polynucleotides of the invention, for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detect presence of the genetic construct in the transformed plant.

Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zin gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.

Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase {bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene ( hpt) for hygromycin resistance. Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336. - · · _. ^■

Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. "Regulatory elements" is used here in the widest possible sense and includes other genes which interact with the gene of interest.

Genetic constructs designed to decrease or silence the expression of a polynucleotide/polypeptide may include an antisense copy of a polynucleotide. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator. ^~

An "antisense" polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,

5'GATCTA 3' (coding strand) 3'CTAGAT 5' (antisense strand)

3'CUAGAU 5' mRNA 5'GAUCUCG 3' antisense RNA Genetic constructs designed for gene silencing may also include an inverted repeat. An 'inverted repeat' is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g., 5'-GATCTA ....TAGATC-3'

3'-CTAGAT ATCTAG-5'

The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually a spacer of at least 3-5 bp between the repeated region is required to allow hairpin formation.

Another silencing approach involves the use of a- small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et al, 2002,. Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated.

The term genetic construct as used herein also includes small antisense RNAs and other such polynucleotides useful for effecting gene silencing.

Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al, 1990, Plant Cell 2, 279; de

Carvalho Niebel et al, 1995, Plant Cell, 7, 347). In some cases sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as ah intron or a 5' or 3' untranslated region (UTR).

Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al, 2002, Plant Physiol. 128(3): 844-53; Jones et al, 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the expression of a sequence operably-linked to promoter of the invention is also contemplated.

The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and/or non-coding sequence, such as promoter and/or intron and/or 5' or 3' UTR sequence, or the corresponding gene. Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (Mclntyre, 1996, Transgenic Res, 5, 257)

Pre-transcriptional silencing may be brought about through mutation of the gene itself or its regulatory elements. Such mutations may include point mutations, frameshifts, insertions, deletions and substitutions.

Plants The term "plant" is intended to include a whole plant or any part of a plant, propagules and progeny of a plant and harvested material from the plant. The harvested material preferably includes plant material processed into feeds.

The term 'progeny' as used herein refers to any cell, plant or part thereof which has been obtained or derived from a cell or transgenic plant of the present invention^'. Thus, the term progeny includes but is not limited to seeds, plants obtained from seeds, plants or parts thereof, or derived from plant tissue culture, or cloning, techniques.

The term 'propagule' means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.

A "transgenic" or transformed" plant refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic ot transformed plant or from a different species. A transformed plant includes a plant which is either stably or transiently transformed with new genetic material.

The plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown. Plants resulting from such standard breeding approaches also form part of the present invention. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the general phenylopropanoid biosynthetic pathway from plants.

Figure 2 shows a Vector NTI map of the construct (MsMYB14pART27) containing the MsMYB14-l genomic sequence genomic allele A from M. sativa, in the binary vector pART27. Figure 3 shows protein alignment of the MYB 14-1 polypeptide sequence (SEQ ID NO: 1) from Medicago sativa and variant polypeptide sequences from Medicago sativa (MsMYB14-2 [SEQ ID NO: 2] and MsMYB14-3 [SEQ ID NO: 3]) and Medicago truncatula (MtMYB14-l [SEQ ID NO: 4] and MtMYB14-2 [SEQ ID NO: 5]). The_. position of the R2 repeat (extending from amino acid 12 [L] to 64 [D]) of the R2R3 MYB DNA binding domain of all five sequences is indicated with arrows. The position of the R3 repeat (extending from amino acid 65 [I] to 1 15[K]) of the R2R3 MYB DNA binding domain of all five sequences is indicated with arrows. The position of the amino acid pair TQ is highlighted with shading at positions 20 and 21 respectively in MtMYB14-l (SEQ ID NO: 4), MsMYB14-2 (SEQ ID NO: 2) and MsMYB14-3 (SEQ ID NO: 3). Figure 4 shows a table with % identity between the MsMYB14-l and variants aligned in Figure

3. ; _. ^' ,

Figure 5 shows expression analysis of MsMYB14-l and variants thereof, The figure shows ethidium bromide stained agarose gels with PCR reactions to test for the presence of MYB 14 from genomic DNA (869 bp) and cDNA (740bp band) isolated from various tissues of Medicago sativa W90-10. Primers were MYBFY (MYB domain) and PMYBR (to 3 'end of gene) amplifying two different sized fragments (approx 869bp genomic fragment containing an intron, and 740bp for cDNA minus the intron).

Lanes on A: Al Ladder; A2 immature seed cDNA, A3 root cDNA, A4 mature leaf cDNA, A5 stem cDNA, A6 Pod cDNA, A7-A1 1 are controls: A7 T.arvense cDNA plasmid control D685, A8 genomic DNA T. arvense control, A9 Medicago sativa W90-10 genomic DNA control D846, A10 T. repens cDNA plasmid control D760, Al 1 water control. Lanes on B: Bl genomic DNA D14 T. repens control, B2 genomic DNA D15 T. arvense control, B3 Medicago sativa AF3010 leaf cDNA, B4 Medicago sativa W90-10 mature seed cDNA, B5 Ladder, B6 water control, B7 Medicago sativa AF3010 flower cDNA, B8 Medicago sativa W90- 10 genomic DNA control, B9 ladder, BIO Medicago sativa AF3010 leaf cDNA, Bl l Medicago sativa AF3010 leaf cDNA, B12 Medicago sativa AF3010 leaf cDNA, B13 Medicago sativa AF3010 leaf cDNA, B14 Medicago sativa AF3010 leaf cDNA, B15 T.arvense cDNA plasmid control D685.

EXAMPLES: Materials and Methods

Plant Material and Analysis of Condensed Tannin Levels

Seeds from several cultivars of the legume species Medicago sativa and Medicago truncatula were grown in glasshouses. Trifolium repens, T. occidentale and T. arvense were also grown for control purposes. Plant material of various ages and types were harvested and the material immediately frozen in liquid nitrogen and subsequently ground and used for isolation of DNA or RNA.

Regenerated tobacco, transformed as described below, plahtlets were eventually transferred into the glasshouses and planted into potting mix when roots had formed.

Isolation of DNA and RNA, and cDNA Synthesis Genomic DNA was isolated from fresh or frozen plant tissues (100 mg) using DNeasy^® Plant

Mini kit (Qiagen) following the manufacturer's instructions. DNA preparations were treated with RNAse H (Sigma) to remove RNA from the samples. Total RNA was isolated from fresh or frozen tissues using RNeasy^® Plant Mini kit (Qiagen). Isolated total RNA (100 μg) was treated with RNAse free DNAse I to remove DNA from the samples during the isolation, following the manufacturer's instructions. Concentration and purity of DNA and RNA samples was assessed by determining the ratio of absorbance at 260 and 280 nm using a NanoDrop ND-100

spectrophotometer. Total RNA (1 μg) was reverse-transcribed into cDNA using SMART™ cDNA Synthesis Kit (Clontech) using the SMART™ CDS primer IIA and SMART II™ A oligonucleotides following manufacturer's instructions. Polymerase chain reaction (PCR) and TOPO cloning of PCR products

Standard PCR reactions were carried out in a Thermal Cycler (Applied Biosystems), a quantity of approximately 5 ng DNA or 1 μΐ cDNA was used as template. The thermal cycle conditions were as follows: Initial reaction at 94°C for 30 sec, 35 cycles at 94°C for 30 sec, 50-64°C for 30 sec (depending on the Tm of the primers), and at 72°C for 1-2 min (1 min/ kb), respectively, and a final reaction at 72°C for 10 min.

PCR products were separated by agarose gel electrophoresis and visualised by ethidium bromide staining. Bands of interest were cut out and DNA subsequently extracted from the gel slice using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer's instructions. Extracted PCR products were cloned into TOPO 2.1 vectors (Invitrogen) and transformed into OneShot® Escherichia, coli cells by chemical transformation following the manufacturer's instructions. Bacteria were subsequently plated onto pre- warmed Luria-Bertani (LB; Invitrogen) agar plates (1% tryptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar) containing 50 μg ml^"1 kanamycin and 40 μΐ of 40 mg ml^"1 X-gal (5-bromo-4-chloro-3-indolyl-X-D-galactopyranoside; Invitrogen) and incubated at 37°C overnight. Positive colonies were selected using white-blue selection in combination with antibiotic selection. Colonies were picked and inoculated into 6 ml LB broth (1% tryptone, 0.5% yeast extract, 1.0% NaCl) containing 50 μg ml^"1 kanamycin and incubated at 37°C in a shaking incubator at 200 rpm.

Bacterial cultures were extracted and purified from LB broth culture using the Qiagen Prep Plasmid Miniprep Kit (Qiagen) following the manufacturer's instructions.

Genetic constructs used in the transformation protocol

The MsMYB14-l of SEQ ID NO: 7 and variants thereof (SEQ ID NOs: 9 and 1 1) were cloned by standard techniques into pART7 downstream of the 35S promoter. The unique Notl fragment was then shuttled into pART27 (Gleave, 1992) for transformation of tobacco and Medicago species. This binary vector contains the nptll selection gene for kanamycin resistance under the control of the CaMV 35S promoter. By way of example, T-DNA of the genetic construct containing the MsMYB14-l gene, showing orientation of cloned gene, is represented graphically in Figure 2. Genetic constructs in pART27 were transferred into Agrobacterium tumefaciens strain GV3101 or EHA105 as plasmid DNA using freeze-thaw transformation method (Ditta et al 1980). The structure of the constructs maintained in Agrobacterium was confirmed by restriction digest of plasmid DNA's prepared from bacterial culture. Agrobacterium cultures were prepared in glycerol and transferred to -80°C for long term storage. Genetic constructs maintained in Agrobacterium strain GV3101 are inoculated into 25 mL of MGL broth containing spectinomycin at a concentration of lOOmg/L. Cultures are grown overnight (16 hours) on a rotary shaker (200rpm) at 28°C. Bacterial cultures are harvested by centrifugation (3000xg, 10 minutes). The supernatant is removed and the cells resuspended in a 5mL solution of lOmM MgS0₄. - Tobacco Transformation

Tobacco can be transformed via the leaf disk transformation-regeneration method (Horsch et all 985). Leaf disks from sterile wild type W38 tobacco plants are inoculated with an Agrobacterium tumefaciens strain containing the appropriate binary vector, and cultured for 3 days. The leaf disks are then transferred to MS selective medium containing 100 mg/L of kanamycin and 300 mg/L of cefotaxime. Shoot regeneration occurs over a month, and the leaf explants are placed on hormone free medium containing kanamycin for root formation. Medicago transformation

M. truncatula and M. sativa transformation can be carried out using a modified method of Wright et al. (2006). Medicago cotyledon explants can be transformed using Agrobacterium tumefaciens AGL-1 (EHA105). Transformed events can be selected using Kanamycin (50mg/L) with cefotaxime. (250 mg/L) and Timentin (300mg/L) to remove the bacterial presence. Seeds are weighed to provide approximately 400 - 500 cotyledons (ie. 200 - 250 seeds) for dissection (0.06gm = 100 seeds). In a centrifuge tube, seeds are subjected to 10ml concentrated H₂S0 for 5-8 minutes, then rinsed three times vvith sterile water. Seeds are then surface sterilised in 30% bleach by shaking on a circular mixer for 8 minutes followed by four washes in sterile water. Seeds are allowed to imbibe overnight at 17°C. Cotyledons are dissected from seeds using a dissecting microscope. Initially, the seed coat and endosperm are removed. Cotyledons are separated from the radical with the scalpel by placing the blade between the cotyledons and slicing through the remaining stalk. Cotyledonary explants are harvested onto a sterile filter disk on TM1 media. For transformation, a 3ul aliquot of Agrobacterium suspension is dispensed on to each dissected cotyledon. Plates are sealed and cultured at 25°C under a 16 hour photoperiod. Following a 48 hour period of co-cultivation, transformed cotyledons are rinsed, then transferred to plates containing TM-2 medium supplemented with 50mg/L Kanamycin, 250 mg/1 cefotaxime, and timentin (300mg/L) and returned to the culture room. Following the regeneration of shoots, explants are transferred to TM-3 medium supplemented with 50mg/L^' Kanamycin, 250 mg/1 cefotaxime and timentin (300mg/L): Regenerating shoots are subcultured every three weeks to fresh TM-3 media containing selection. As root formation occurs, plantlets are transferred into tubs containing MSO medium containing kanamycin selection. Large clumps of regenerants are divided to individual plantlets at this stage. Whole, rooted plants growing under selection are then potted into sterile peat plugs.

DNA Sequencing

Isolated plasmid DNA was sequenced using the dideoxynucleotide chain termination method, using Big-Dye (Version 3.1) chemistry (Applied Biosystems). Either Ml 3 forward and reverse primers or specific gene primers were used. The products were separated on an ABI Prism 3100 Genetic Analyser (Applied Biosystems) and sequence data were compared with sequence information published in GenBank (NCBI) using AlignX (Invitrogen).

DMACA staining of plant material

CTs can be histochemically analysed using the acidified DMACA (4-dimethylamino- cinnamaldehyde) method essentially as described by Li et al. (1996). This method uses the

DMACA (p-dimethylaminocinnamaldehyde) reagent as a rapid histochemical stain that allows specific screening of plant material for very low CT accumulation. The DMACA-HC1 protocol is highly specific for proanthocyanidins. This method is preferred to the vanillin test as anthocyanins seriously interfere with the vanillin assay. Tissues of various ages may be sampled and tested.

LCMSMS Methodology for HPLC analysis

To extract flavonoids for HPLC analysis, plant tissue, such as leaf tissue (0.5 g fresh weight) is frozen in liquid N_2; ground to a fine powder and extracted with acetic acid: methanol (80:20 v/v) for 30 mins at 4°C. The plant debris is pelleted in a microcentrifuge at 13K rpm for lOmins. The supernatant is removed and placed at -20°C for 30 mins. An aliquot is used for HPLC analysis. An aliquot is analysed by HPLC using both UV-PDA and MS/MS detection on a Thermo LTQ Ion Trap Mass Spectrometer System. ' The extracts are resolved on a Phenomonex Luna CI 8 reversed phase column by gradient elution with water and acetonitrile with 0.1% formic acid as the mobile phase system. Detection of the anthocyanins is by UV absorption at 550nm, and the other metabolites are estimated by either MSI or MS2 detection by the mass spectrometer.

The instrument used can be a linear ion trap mass spectrometer (Thermo LTQ) coupled to a Thermo Finnigan Surveyor HPLC system .(both San Jose, CA, USA) equipped with a Thermo photo diode array (PDA) detector. Thermo Finnigan Xcalibur software (version 2.0) can be used for data acquisition and processing. A 5 aliquot of sample is injected onto a 150x2. lmm Luna C18(2) column (Phenomenex, Torrance, CA) held at a constant 25 °C. The HPLC solvents used are: solvent A = 0.1 % formic acid in H₂0; solvent B - 0.1% formic acid in Acetonitrile. The flow rate is 200 μΤ min^"1 and the solvent gradient used is shown in Table 1 below. PDA data is collected across the range of 220nm-600 nm for the entire chromatogram.

Time (min) Solvent A% Solvent B%

0 95 5

6 95 5

11 90 10

26 83 17

Table 1 : HPLC gradient.

The mass spectrometer is set for electrospray ionisation in positive mode. The spray voltage was 4.5 kV and the capillary temperature 275°C, and flow rates of sheath gas, auxiliary gas, and sweep gas are set (in arbitrary units/min) to 20, 10, and 5, respectively. The first 4 and last 1 1 minutes of flow from the HPLC are diverted to waste. The MS is programmed to scan from 150-

1 3 1

2000 m/z (MS scan), then perform data dependant MS on the most intense MS ion. The isolation windows for the data dependant MS³ method is 2 mu (nominal mass units) and fragmentation (35% CE (relative collision energy)) of the most intense ion from the MS¹ spectrum is followed by the isolation (2 mu) and fragmentation (35% CE) of the most intense ion from the MS spectrum. The mass spectrometer then sequentially performs selected reaction monitoring (SRM) on the masses in Table 2 below, with isolation windows for each SRM of 2.5 mu and fragmentation CE of 35%. These masses listed cover the different combinations of procyanidin (catechin and/or epicatechin) and prodelphinidin (gallocatechin or epigallocatechin) masses up to trimer.

595.3 160-2000 PC:PD dimers

61 1.3 165-2000 PD:PD dimers

867.3 235-2000 PC:PC:PC timers

883.3 240-2000 PC:PC:PD trimers

899.3 245-2000 PC:PD:PD trimers

915.3 250-2000 PD:PD:PD trimers

Table 2: SRM masses for monomers, dimers and trimers:

Results

Example 1: Identification of the MsMYB14 and MtMYB14 polynucleotides and polypeptides of the invention The applicants isolated the gene represented in SEQ ID NO: 7 from Medicago sativa. The applicants designated this gene MsMYB14-l . The open reading frame/coding sequence of MsMYB14-l is shown in SEQ ID NO: 6. The encoded MsMYB14-l protein is shown in SEQ ID NO: 1.

The applicants also identified and isolated variants (polynucleotide and polypeptide) of

MsMYB14-l from a range of accessions of Medicago sativa (MsMYB 14.-2, MsMYB14-3) and M. truncatula, (MtMYB14-l , MtMYB14-2) as summarized in the Summary of Sequences table below.

An alignment of the MsMYB 14-1 polypeptide sequence and variant sequences (MsMYB 14-2, MsMYB 14-3, MtMYB 14-1 , MtMYB 14-2) is shown in Figure 3. These sequences have the structure of R2R3 MYB transcription factors (Stracke et al., 2001). The position of the R2 and R3 repeats in the MYB DNA binding domain are shown in Figure 3. The remainder of the molecules from the C terminus of the R3 repeat to the C terminus represents the acid, or activation, domain;

Percent identity between the polypeptide sequences is shown in Figure 4. The fully integrated software package available in the Vector NTI programs (available from www.invitrogen.com), was used for all analyses, including alignments that are based on a ClustalW algorithm.

This alignment shows which amino acids are conserved between MsMYB14-l and all of the variants, and which amino acids are varied.

The applicants identified two sequence motifs that are highly conserved between MsMYB14-l and all of the variants. These are shown in SEQ ID NO:22 and 23. The position of the motifs is highlighted in grey on Figure 4. The applicants assert that the presence of either or preferably both motifs is indicative of function in controlling CT production. The applicants also identified the presence of a TQ (threonine gluatamine). amino acid pair present in the R2 repeat of the R2R3 MYB DNA binding domain of the polypeptide sequences of MtMYB 14-1 (SEQ ID NO:4), MsMYB 14-2 (SEQ ID NO:2) and MsMYB14-3 (SEQ ID NO:3). The TQ is present at amino acid positions 20 and 21 respectively in each of the three sequences and is highlighted with shading in Figure 3. This amino acid pair was also found in the same position in a clover R2R3 MYB sequence shown by the applicants to be capable of inducing production of flavonoids, particularly condensed tannins, in plants. This provides evidence of the significance of the TQ amino acid pair for inducing production of flavonoids, particularly condensed tannins, in plants.

The significance of these amino acids can be further confirmed by altering these amino acids (for example by site directed mutagenesis methods well-known to those skilled in the art) and testing the function of the altered transcription factors for inducing flavonoid, particularly condensed tannin production, in plants following the methodology described in Example-3 below.

Example 2: Expression of MsMYB14 correlates with accumulation of condensed tannins in plants

Seeds from a number of accessions representing various genotypes from two Medicago species, Medicago sativa and Medicago trancatula, were grown in a glasshouse.

Analysis of CT accumulation Presence or absence of CTs was determined in leaves using DMACA staining as described in the Materials and Methods section. CTs were only detectable in Medicago sativa (using DMACA staining) in the leaf trichomes on the.abaxial epidermal surface and in the seed coats.

Analysis of MYB 14 transcript accumulation Primers for amplifying MsMYB14-l , or variants thereof, were designed and transcript levels in various tissues were determined by PCR.

Primer used to amplify the MYB 14 sequences are listed below. These primers allowed the isolation of the alleles as a single fragment, or as two overlapping fragments.

RNA was isolated from leaves, roots, pods, flowers and seeds of two Medicago sativa accessions (W90-10 and AF3010) and converted to cDNA. PCR reactions were performed using the prepared cDNA with two primers designed to amplify the MsMYB14-l or variants thereof, giving different sized bands to allow distinction between the mRNA and genomic allele amplification. Genomic DNA and cloned cDNA from a number of species were included for controls. Trifolium tissues are included as control because there is a sequence closely related to MsMYB14 (unpublished), in Trifolium.

Transcript analysis revealed that the MsMYB14-l gene (and variants thereof) was expressed only in tissues actively accumulating CTs; namely the developing seed, but was not detected in mature or emergent leaf tissue, stems, internodes, roots, and petioles. PCR results of the analysis are shown in Figure 5.

Thus expression of MsMYB14-l and variants thereof is strongly correlated with accumulation of CTs. Example 3: Transformation of plants with the MsMYB14-l sequence of the invention in order to alter condensed tannins content

Genetic constructs for plant transformation

The plant transformation vectors pART7 and binary vector pART27 (Gleave 1992) were used for the genetic constructs. This binary vector contains the nptll selection gene for kanamycin resistance under the control of the CaMV 35S promoter. Cloning of expression cassettes into the binary vector pART27, is facilitated by a unique Notl restriction site and selection of recombinants by blue/white screening for β-galactosidase. The MsMYB 14-1 sequence of SEQ ID NO: 7 was cloned as an EcoRI fragment into pART7 so that it could be expressed from the CaMV 35S promoter. The constructs were then shuttled to pART27 as a Notl fragment to produce MsMYB 14pART27. A schematic representation of MsMYB 14pART27, showing orientation of cloned gene, is provided in Figure 2. The sequence of MsMYB 14pART27 is shown in SEQ ID NO: 16. Similar constructs can of course be used to demonstrate the function of variants of MsMYB 14-1 described herein.

Genetic constructs in pART27 were transferred into Agrobacterium tumefaciens strain GV3101 or EHA105 as plasmid DNA using freeze-thaw transformation method (Ditta et al 1980). The structure of the constructs maintained in Agrobacterium was confirmed by restriction digest of plasmid DNA's prepared from bacterial culture. Agrobacterium cultures were prepared in glycerol and transferred to -80°C for long term storage. Genetic constructs maintained in Agrobacterium are inoculated into 25 mL of MGL broth containing spectinomycin at a concentration of l OOmg/L. Cultures are grown overnight (16 hours) on a rotary shaker (200rpm) at 28°C. Bacterial cultures are harvested by centrifugation (3000xg, 10 minutes). The supernatant is removed and the cells resuspended in a 5mL solution of lOmM MgS0₄ or TM-1 medium.

Transformation of Medicago sativa M. sativa was transformed as described in the Materials and Methods section, using binary vector MsMYB 14 pART27.

Transformation of tobacco

Tobacco plants were also transformed with MsMYB 14pART27 as described in the Materials and Methods section.

To test for integration of the MsMYB 14-1 transgene

DNA was extracted from transgenic plants to test for integration of the MsM14pART27 binary vector.

PCR reactions can be performed using primer sets designed by standard procedures to amplify a portion of the inserted transgene, such as for example the 35S promoter and the majority of the transformed gene, by standard techniques, and as described in the Materials and Methods section, to confirm integration of the transgene into the plant genome.

Tobacco DMACA Analysis

To test for production of condensed tannins in the transformed tobacco plants DMACA analysis can be performed, as described in the Materials and Methods section, on tissue excised from transgenic plants.

Transgenic plant tissues should be compared to tissues from control plants," such as non- transformed plants or plants transformed with empty (insert-free) constructs.

A suitable tissue to compare is leaf tissue. CTs may be detected at a higher level in tissues of transgenic tobacco expressing MsMYB 14-1 or a variant thereof, relative to wild type or untransformed tobacco, and/or may be present in tissues of transgenic plants but absent in control plant tissues that do not accumulate CT, such as vegetative tissues.

Such experiments could confirm that the M. sativa MYB14-1 gene is able to alter condensed tannin production in plants transgenic.

Similar transformation, DMACA analysis and HPLC/LCMS analysis experiments can be performed to confirm the function of the variant sequences (MsMYB 14-2, MsMYB 14-3, MtMYB14-l , MtMYB14-2) as summarized in the Brief Description of the Figures and Sequence Listing.

Tobacco HPLC/LCMS Analysis

HPLC/LCMS analysis may also be performed on MsMYB 14-1 transgenic tobacco and control plant tissues. Flavonoids should be extracted from transgenic and control tobacco plants, and processed via HPLC as described in the Materials and Methods section. Results of these analyses may be used to confirm the presence of CT in tissues of transgenic tobacco samples versus absence or lower levels of CTs in control plant tissue samples.

Medicago DMACA Analysis Production of CT can be assessed using DMACA staining as described in the Materials and Methods section. The CT specific stain, DMACA, stains tissues of plants accumulating CTs, more highly than those not accumulating CTs.

Comprising transgenic and control plants as described for tobacco above can be used to confirm the function of the MsM YB 14-1 sequence in increasing CT production. Medicago HPLC/LCMS Analysis

HPLC/LCMS analysis may also be performed for MsMYB 14-1 transgenic Medicago, as described above and in the Materials and Methods section.

Confirming the function of variant sequences

Similar transformation, DMACA analysis and HPLC/LCMS analysis experiments can be performed to confirm the function of the variant sequences (MsMYB 14-2, MsMYB 14-3, MtMYB14-l, MtMYB14-2 as summarized in the Brief Description of the Figures and Sequence Listing).

Use of tobacco transformation combined with DMACA analysis is a particularly quick and simple method for confirming the function of variant sequences in production of CTs. Constructs including the cDNA or genomic sequences of the variants may be used in transformation experiments, using techniques described above and/or well known to those skilled in the art. REFERENCES:

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Dixon RA, Xie DY, Sharma SB (2005) .Proanthocyanidins— a final frontier in flavonoid research? New Phytologist 165: 9-28. Douglas GB, Wang Y, Waghorn GC, Barry TN, Purchas RW, Foote AG, Wilson GF (1995). Liveweight Gain And Wool Production Of Sheep Grazing Lotus-Corniculatus And Lucerne (Medicago-Sativa). New Zealand Journal Of Agricultural Research 38: 95-104. Gleave AP (1992). A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Molecular Biology 20: 1203-1207.

Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT. (1985). A simple and general method for transferring genes into plants. Science. ;227: 1229-1231. Kingston- Smith AH, Thomas HM (2003). Strategies of plant breeding for improved rumen function Annals of Applied Biology 142:13-24.

McKenna, P.B (1994). The occurrence of anthelminitic resistant sheep nematodes in the southern North Island of New Zealand. NZ Veterinary. Journal. 42: 151-152.

McMahon LR, McAllister TA, Berg BP, Majak W, Acharya SN, Popp JD, Coulman BE, Wang Y, Cheng KJ (2000). A review of the effects of forage condensed tannins on ruminal

fermentation and bloat in grazing cattle. Canadian Journal of Plant Science 80: 469-485.

Marten, G. C, Ehle, F.R. & Ristau, E. A. (1987). Performance and photosensitization of cattle related to forage quality of four legumes. Crop Science 27: 138-145.

Molan. A.L. Waghorn, G.C., McNabb, W.C. (2001). Effect of condensed tannins on egg hatching and larval development of Trichostrongylus colobriformis in vitro. The Veterinary Record 150: 65-69.

Niezen, J.H., Waghorn, T.S., Waghorn, G.C. and Charleston, W.A.G. (1993) Internal parasites and lamb production - a role for plants containing condensed tannins. Proc. NZL. Soc. Anim. Prod. 53, pp. 235-238. Puchala, R., Min, B. R., Goetsch, A. L. and Sahlu, .T. (2005). The effect of a condensed tannin- containing forage on methane emission by goats. Journal of . Animal Science 83:182-186.

Rumbaugh, M.D. (1985). Breeding bloat-safe cultivars of bloat-causing legumes. In: Barnes,

R.F., Ball, P.R., Bringham, R.W., Martin, G.C, Minson, D.J. (Eds.), Forage Legumes for Energy-Efficient Animal Production. USDA, Washington. Proc. Bilateral Workshop, Palmerston North, NZ, April 1984, pp. 238-245.

Small, E. & M. Jomphe. 1989. A synopsis of the genus Medicago (Leguminosae). Canad. J. Bot. 67:3260-3294. -

Stracke, Ralf, Werber Martin, and Bernd Weisshaar. (2001) The R2R3-MYB gene family in Arabidopsis thaliana. Curr. Opin. Plant Biol. 4, 447^-56.

Sykes. A.R and Coop. R.L (2001). Interaction between nutrition and gastrointestinal parasitism in sheep New Zealand Veterinary Journal. 49: 222-226. Tanner GJ, Moore AE, Larkin PJ (1994). Proanthocyanidins Inhibit Hydrolysis Of Leaf Proteins By Rumen Microflora In- Vitro British Journal Of Nutrition 71 : 947-958.

Waghorn, G.C., Douglas, G.B.,Niezen, J.H., McNabb, W.C and Foote, A.G (1998). Forages with condensed tannins- their management and nutritive value for ruminants. Proceedings of the New Zealand Grasslands Association 60: 89-98. Wright E. Dixon R, Wang Z-Y (2006). Medicago truncatula transformation using cotyledon explants. in Methods in Molecular Biology/. Agrobacterium protocols. Second ed, Volume 1 editor: Kan Wang. Humana Press.

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SUMMARY OF SEQUENCES

SEQ ID NO Type Species Reference

1 Polypeptide Medicago sativa MsMYB14-l

2 Polypeptide Medicago sativa MsMYB14-2

3 Polypeptide Medicago sativa MsMYB14-3

4 Polypeptide Medicago truncatula MtMYB14-l

5 Polypeptide Medicago truncatula MtMYB14-2

6 Polynucleotide Medicago sativa MsMYB14-l cDNA

7 Polynucleotide Medicago sativa MsMYB14-l gDNA

8 Polynucleotide Medicago sativa MsMYB14-2 cDNA

9 Polynucleotide Medicago sativa MsMYB14-2 gDNA (W10)

10 Polynucleotide Medicago sativa MsMYB14-3 cDNA

1 1 Polynucleotide Medicago sativa MsMYB14-3 gDNA (W7)

12 Polynucleotide Medicago truncatula MtMYB14-l cDNA

13 Polynucleotide Medicago truncatula MtMYB14-l gDNA (Y10)

14 Polynucleotide Medicago truncatula MtMYB14-2 cDNA

15 Polynucleotide Medicago truncatula MtMYB14-2 gDNA (Y4)

16 Polynucleotide Vector MsMYBpART27

17 Polynucleotide Artificial, primer MYB14 ATG Polynucleotide Artificial, primer MYB14 TGA

Polynucleotide Artificial, primer MYB14 MsatR'

Polynucleotide Artificial, primer MYB14 MsatF

Polynucleotide Artificial, primer MYB STF

Polypeptide Artificial Conserved motif 1

Polypeptide Artificial Conserved motif 2

Claims

CLAIMS:

I . An isolated polynucleotide encoding a polypeptide comprising the amino acid sequence of , any one of SEQ ID NO: 1 to 5 or a variant or fragment thereof.

2. The isolated polynucleotide of claim 1 wherein the polypeptide is an R2R3 MYB

transcription factor.

3. The isolated polynucleotide of claim 2 wherein the R2R3 MYB transcription factor contains the amino acid pair TQ (threonine glutamate) in the R2 repeat of the R2R3 DNA binding domain.

4. The isolated polynucleotide of claim 3 wherein the T and Q are at amino acid positions 20 and 21 respectively in the R2R3 MYB transcription factor polypeptide.

5. The isolated polynucleotide of any one of claims 1 to 4 wherein the polypeptide is capable of positively regulating production of at least one flavonoid in a plant.

6. The isolated polynucleotide of claim 5 wherein the flavonoid is a condensed tannin.

7. The isolated polynucleotide of any one of claims 1 to 6 wherein the variant has at least 70% identity to the amino acid sequence of any one of SEQ ID NO: 1 to 5.

8. The isolated polynucleotide of any one of claims 1 to 7 wherein the variant comprises the amino acid sequence of SEQ ID NO:22

9. The isolated polynucleotide of any one of claims 1 to 7 wherein the variant comprises the amino acid sequence of SEQ ID NO:23

10. The isolated polynucleotide of any one of claims 1 to 7 wherein the variant comprises the amino acid sequence of both SEQ ID NO:22 and SEQ ID NO:23

I I . The isolated polynucleotide of any one of claims 1 to 10 wherein the variant is derived from a Medicago species. -

12. The isolated polynucleotide of any one of claims 1 to 1 1 wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1 to 5.

13. An isolated polynucleotide comprising the sequence of any one of SEQ ID NO: 6 to 15 or a variant or fragment thereof.

14. The isolated polynucleotide of claim 13 that encodes an R2R3 MYB transcription factor.

15. The isolated polynucleotide of claims 14 wherein the R2R3 MYB transcription factor contains the amino acid pair TQ (threonine glutamate) in the R2 repeat of the R2R3 DNA binding domain.

16. The isolated polynucleotide of claim 15 wherein the T and Q are at amino acid positions 20 and 21 respectively in the R2R3 MYB transcription factor polypeptide.

17. The isolated polynucleotide of any one of claims 13 to 16 that encodes a polypeptide that is capable of positively regulating production of at least one flavonoid in a plant.

18. The isolated polynucleotide of claim 17 wherein the flavonoid is a condensed tannin.

19. The isolated polynucleotide of any one of claims 13 to 18 wherein the variant has at least 70% identity to the sequence of any one of SEQ ID NO: 6 to 15.

20. The isolated polynucleotide of any one of claims 13 to 19 wherein the variant is derived from a Medicago species.

21. The isolated polynucleotide of any one of claims 13 to 20 wherein the polynucleotide comprises the sequence of any one of SEQ ID NO: 6 to 15.

22. An isolated polypeptide comprising the amino acid sequence of any one of SEQ ID NO: 1 to 5 or a variant or fragment thereof. -

23. The isolated polypeptide of claim 22 wherein the variant is an R2R3 MYB transcription factor.

24. The isolated polypeptide of claim 23 wherein the R2R3 MYB transcription factor contains the amino acid pair TQ (threonine glutamate) in the R2 repeat of the R2R3 DNA binding domain.

25. The isolated polypeptide of claim 24 wherein the T and Q are at amino acid positions 20 and 21 respectively in the R2R3 MYB transcription factor polypeptide.

26. The isolated polypeptide of any one of claims 22 to 25 wherein the polypeptide is capable of positively regulating production of at least one flavonoid in a plant.

27. The isolated polypeptide of claim 26 wherein the flavonoid is a condensed tannin.

28. The isolated polypeptide of any one of claims 22 to 27 wherein the variant has at least 70% identity to the amino acid sequence of any one of SEQ ID NO : 1 to 5.

29. The isolated polypeptide of any one of claims 22 to 28 wherein the variant comprises the amino acid sequence of SEQ ID NO:22

30. The isolated polypeptide of any one of claims 22 to 28 wherein the variant comprises the amino acid sequence of SEQ ID NO:23

31. The isolated polypeptide of any one of claims 22 to 28 wherein the variant comprises the amino acid sequence of both SEQ ID NO:22 and SEQ ID NO:23

32. The isolated polypeptide of any one of claims 22 to 31 wherein the variant is derived from a Medicago species.

33. The isolated polypeptide of any one of claims 22 to 32 wherein the polypeptide comprises the amino acid sequence of any one of SEQ ID NO: 1 to 5.

34. An antibody raised against the polypeptide of any one of claims 22 to 33.

35. An isolated polynucleotide encoding a polypeptide of any one of claims 22 to 33.

36. An isolated polynucleotide comprising:

a) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of a polynucleotide of any one of claims 1 to 21 and 35;

b) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of any one of claims 1 to 21 and 35; or

d) a polynucleotide comprising a sequence, of at least J 5 nucleotides in length, capable of hybridising to the polynucleotide of any one of claims 1 to 21 and 35.

37. A genetic construct, expression construct, or RNAi construct comprising a polynucleotide of any one of claims 1 to 21, 35 and 36.

38. A host cell genetically modified to express a polynucleotide of any one of claims 1 to 21, 3_.5 and 36, or a polypeptide of any one of claims 22 to 33.

39. A host cell comprising a genetic construct, expression construct, or RNAi construct of claim ■ 37.

40. The host cell of claim 39 that is a plant cell.

■

41. A plant comprising the plant cell of claim 40.

42. A plant genetically modified to express a polynucleotide of any one of claims 1 to 21 , 35 and 26, or a polypeptide of any one of claims 22 to 33.

43. A plant comprising a genetic construct, expression construct, or RNAi construct of claim 37.

44. A method for producing a plant cell or plant with altered flavonoid content, the method comprising transformation of a plant cell, or plant, with a polynucleotide of any one of claims 1 to 21 , 35 and 36.

The method of claim 44 wherein the flavonoid content is increased in the plant produced.

46. The method of claim 44 or 45 wherein the flavonoid is produced in a foliar tissue.

47. The method of any one of claims 44 to 46 wherein the flavonoid is produced in a leaf.

48. The method of any one of claims 40 to 47 wherein flavonoid is produced in an epidermal tissue.

49. The method of any one of claims 40 to 48 wherein the flavonoid is produced in a tissue, or cell type, that is substantially devoid of the flavonoid, in a control plant, such as a similar plant. that has not been transformed.

50. The method of any one of claims 40 to 49 wherein the flavonoid is a condensed tannin.

51. A method for producing a flavonoid, the method comprising culturing a host cell, comprising a genetic construct or expression construct of claim 37, under conditions in which the polynucleotide of any one of claims 1 to 21, 35 and 36, or b) the polypeptide of any one of claims 22 to 33, is expressed in the cell, leading to production of the flavonoid in the cell.

52. The method of claim 51 in which the host cell is a plant cell.

53. The method of claim 52 in which the plant cell is part of a plant.

54. The method of any one of claims 51 to 53 in which the flavonoid is a condensed tannin.

55. A plant produced by a method of any one of claims 42 to 50, 53 and 54.

56. A plant part, propagule, seed, fruit, progeny, or harvested material derived from the plant of any one of claims 41 to 43, and 55.

57. The plant part, propagule, seed, fruit, progeny, or harvested material of claim 56 that is genetically modified to comprise at least one of:

a) the polynucleotide of any one of claims 1 to 21 , 35 and 36,

b) the polypeptide of any one of claims 22 to 33, and

c) the genetic construct, expression construct, or R Ai construct of claim 35.

58. A method for selecting a plant with altered flavonoid content, the method comprising testing of a plant for altered expression of a polynucleotide of any one of claims 1 to 21 , 35 and 36, or a polypeptide of any one of claims 22 to 33.

59. A group or population of plants selected by the method of claim 58.