AU671272B2 - Regulation of plant genes - Google Patents

Description

OPI DATE 03/08/93 AOJP DATE 14/10/93 APPLN. ID 36454/93 11 PCT NUMBER PCT/GB93/00019 I1111I iii l11111111 l llll ll 11ll III AU9336454 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 93/14211 C12N 15/82, 15/29, C12Q 1/68 Al A01H 5/00, 5/02 (43) International Publication Date: 22 July 1993 (22.07.93) (21) International Application Number: (22) International Filing Date: Priority data: 818,570 9 January Parent Application or Grant (63) Related by Continuation

US

Filed on PCT/GB93/00019 8 January 1993 (08.01.93) 1992 (09.01.92) FRE-I)MAN, Roger IGB/GHI; Diatech Limited, 19 Millers Yard, 10/11 Mill Lane, Cambridge CB2 IRO

(GB).

(74) Agents: MARCHANT, James, lan et al.; Elkington and Fife, Prospect House, 8 Pembroke Road, Sevenoaks, Kent TN13 IXR (GB).

(81) Designated States: AU, JP, US. European patent (AT, BE, CH, DE, DK, ES, FR, GB, GR, IT, LU, MC, NL, PT, SE).

Published With international search report.

Before the expiration of the time limit for amending the claims and to be republished in the event of the receipt of amendments.

818,570 (CIP) 9 January 1992 (09.01.92)

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"f :t)(72)App~;ieentaoe-Inventors: COEN, Enrico [GB/GB]; CARPENTER, Rosemary [GB/GB]; GOODRICH, Justin [GB/GB]; John Innes Institute, Colney Lane, Norwich, Norfolk NR4 7UN (GB).

lo R/Ao /9 0AJ 7/2 0J//,J S ,"Ss-r7" Coy- uic, 7 ,,UATO 7u// (54) Title: REGULATION OF PLANT GENES (57) Abstract A method of regulating the expression of one or more anthocyanin pigment genes in a plant which comprises the steps of transforming plant tissue with an expression vector comprising a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which shows substantial homology with the DEL protein as shown in SEQ ID NO I or 2 and which is capable of regulating expression of one or more plant genes involved in pigment biosynthesis, the said DNA segment being under the control of a promoter upstream of and operably linked thereto and regenerating from the transformed tissue plants showing altered anthocyanin pigmentation.

67 127 J WO 93/14211 PCT/GB93/00019 REGULATION OF PLANT GENES This invention relates to the regulation of plant genes, more. particularly the regulation of genes which control the pigmentation of plants.

Technological Background.

Plant species display remarkable diversity in the pattern and intensity of their pigmentation, in particular red or purple anthocyanin pigmentation. Mutations that block anthocyanin production are viable and have readily observable phenotypes; consequently two classes of genes affecting anthocyanin biosynthesis have been characterised in a range of species including maize, Antirrhinum majus, pea and Petunia hybrida (see Coe and Nuffer in Corn and corn improvement, ed. Sprague, 19-53, American Society of Agronomy, hadison, Wisconsin (1977), Dooner et al, Ann.

Rev. Genet., 25, 173-199 (1992), Martin et al, Soc. Exp.

Biol. Symp., 32, 19-52 (1987), Harker et al, The Plant Cell, 2, 185-194 (1990) and Gerats and Martin, Recent Advances in Phytochemistry, H. Stafford, Ed., (in press).

The genes of one class encode enzymes required for pigment biosynthesis and many of these genes appear to be common to different species (see Martin et al, The Plant Journal, 1, 37-49 (1991), Sommer and Saedler, Mol. Gen.

Genet., 202, 429-434 (1986), Coen et al, Cell, 47, 285-296 (1986) and Beld et al, Plant Mol. Biol., 13, 491-502 (1989)). The other class comprises regulators of the biosynthetic genes (see Almeida et al, Genes Dev., 3, 1758- 1767 (1989), Harker et al ibid, Dooner and Nelson Genetics, 91, 309-315 (1979) and Beld et al ibid) This class includes the C1 and R genes in maize, which encode products related to the myb and myc families of transcription factors respectively (see Paz-Ares et al, EMBO 6, 3553- 3558 (1987) and Ludwig et al, Proc. Natl. Acad. Sci.

WO 93/14211 PCf G/B93/00019 86, 7092-7096 (1989)).

The present invention is based on the isolation and characterisation of a gene designated delila that regulates pigmentation pattern in Antirrhinum majus and the use of this gene to regulate the expression of one or more anthocyanin pigment genes in a plant. Wild-type A. majus flowers have five red petals united to form a corolla tube with five distinct lobes. The epidermal cells of the petals contain red anthocyanin pigments. A recessive delila (del) mutation is known which confers a strikingly different pattern of floral pigmentation in which the corolla tubes are ivory and the lobes fully pigmented. The del mutation also blocks pigmentation of the anther filaments and lower stems and reduces that of the styles, sepals, carpels and petioles (leaf stalks). The wild-type del product is required in the corolla tube for normal transcript levels of many of the anthocyanin biosynthetic genes (see Almeida et al, ibid and Martin et al ibid).

Although pigmentation of the corolla lobes is normally unaffected by del, in certain genetic backgrounds an effect of del in the lobes is revealed suggesting that del can also act in lobes.

As described in more detail below, the del locus of A.

majus has now been cloned by a method involving transposon tagging and has been found to encode a potential protein (DEL) of 644 amino acids. The cDNA sequence of the cloned del locus and the deduced amino acid sequence of the DEL protein are shown in SEQ ID NO 1 and the deduced amino acid sequence of the DEL protein is shown in SEQ ID NO 2.

Summary of the Invention.

According to one aspect, the present invention provides a method for regulating the expression of one or more anthocyanin pigment genes in a plant which comprises the steps of transforming plant tissue with an expression vector comprising a DNA segment encoding a protein having 'WO 93/14211 PCT/GB93/00019 the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequer-e which shows substantial homology with the DEL protein cshown in SEQ ID NO 1 or 2 and which is capable of regulating expression of one or more plant genes involved in pigment biosynthesis, the said DNA segment being under the control of a promoter upstream of and operably linked thereto and regenerating from the transformed tissue plants showing altered anthocyanin pigmentation. Preferably the DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 is a protein having an amino acid sequence which is at least preferably at least 90%, more preferably at least 98% similar with the DEL protein as shown in SEQ ID NO 1 or 2.

According to another aspect, the present invention provides a plant having a DNA segment as defined above incorporated into its genome or plant propagation material (such as seeds) of such a plant.

According to a still further aspect, the present invention provides a DNA molecule encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which is, at least 80%, preferably at least 90%, more preferably at least 98% similar with the DEL protein as shown in SEQ ID NO 1 or 2 with the DEL protein as shown in SEQ ID NO 1 or 2 and which is capable of regulating expression of one or more plant genes involved in pigment biosynthesis.

According to a still further aspect the present invention provides the use of the DNA molecule encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO's 1 or 2 or a protein having an amino acid sequence which is at least 80%, preferably at least more preferably at least 98% similar with the DEL protein as shown in SEQ ID NO 1 or 2 or the protein encoded thereby to isolate a DNA molecule encoding a protein having the amino acid sequence which shows substantial homology ,WO 93/14211 PCT/GB93/00019 with the DEL protein as shown in SEQ ID NO 1 or 2 from other plant species.

According to a still further aspect the present invention provides an expression vector comprising a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which is at least preferably at least 90%, more preferably at least 98% similar with the DEL protein as shown in SEQ ID NO 1 or 2 and which is capable of regulating expression of one or more plant genes involved in pigment biosynthesis, the said DNA segment being under the control of a promoter upstream of and operably linked thereto. The invention also provides a protein which is the product of expression of the expression vector as defined above in a host cell.

According to a still further aspect, the present invention provides a construct which comprises a transposon having cloned therein a DNA segment as defined above, the said DNA segment being under the control of a minimal promoter upstream of and operably linked thereto.

According to a still further aspect of the invention the present invention provides a method of trapping a promoter/enhancer which comprises the steps of introducing the construct into plant by transformation and propagating from said plant, plants having a phenotype showing altered anthocyanin pigmentation arising as a consequence of transposition of the construct.

According to a still further aspect of the invention the present invention provides a method for isolating a trapped promoter/enhancer from a plant which has been transformed with the construct as defined above which comprises reisolating the construct from said plant together with sequences adjacent thereto.

According to a still further aspect of the invention the present invention provides a method of expressing a gene of interest in a plant, which comprises transforming a cell of said plant with a first construct having said PCT/GB 93 /00019 14 FEBRUARY 1994 gene of interest under the control of a first promoter, which first promoter is that of an anthocyanin gene, upstream of and operably linked thereto, the said plant having incorporated into its genome a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which is at least 80%, preferably at least more preferably at least 98% homologous with the DEL protein as shown in SEQ ID NO 1 or 2 under the control of a second promoter upstream of and operably linked thereto, or the said plant being co-transformed with a second construct which comprises said DNA segment under the control of a third promoter, which third promoter may be the same or different to the second promoter, upstream of and operably linked thereto, or the said first construct optionally including the said DNA segment under the control of said second or third promoter upstream and operably linked thereto if the said plant does not have in its genome the said DNA segment or is not co-transformed with the said second construct, and deriving from the transformed plant further plants expressing said gene of interest.

According to a further aspect of the invention the present invention provides a method of expressing a gene of interest in a plant which comprises transforming said plant with a construct, which construct comprises a transposon having cloned therein a DNA segment as defined above, the said DNA segment being under the control of a minimal promoter upstream of and operably linked thereto, deriving from the transformed plant further plants having a phenotype showing altered anthocyanin pigmentation, reisolating from said plant the said construct together with sequences adjacent thereto, replacing said DNA segment in said construct with a gene of interest to form a new construct and transforming said plant with said new construct.

tT. irrur 2ET I. i~ :~3tM~i;l~,TITU T' "-HIE E~ WO 93/14211 PCr/GB93/00019 The cDNA encoding the DEL protein as shown in SEQ ID NO 1 or 2 contains a long open reading frame (ORF) starting at position +25. The ORF encodes a potential protein, DEL, of 644 amino acids which shows strong homology to the products of .Lc and R-S, two members of the R gene family which controls pigmentation pattern in maize.

Maize and Antirrhinum are taxonomically distant and belong to the monocotyledoneae and the dicotyledoneae respectively, two groups thought to have diverged about 200 million years ago at an early stage in the evolution of flowering plants. There are marked differences in morphology and pigmentation pattern between the two species. The flowers of Antirrhinum are pollinated by bees and have large, vividly pigmented petals. In maize, which' is wind-pollinated, the flowers are inconspicuous and there is no organ with obvious homology to petals. The organ most commonly pigmented is the seed, although the diverse alleles of the R gene family can pigment most plant tissues.

The structural and functional homology between the DEL protein and the prote 4 ns encoded by the R gene family of maize strongly suggests that the control of pigmentation pattern is mediated by a common regulator in different species, in spite of wide differences in morphology and coloration. Accordingly the present invention is not confined to the regulation of the expression of anthocyanin pigments in Antirrhinum but extends generally to all plants where a protein homologous to the DEL protein has an effect on the expression of anthocyanin pigments. The plant preferably belongs to the dicotyledoneae.

The manner in which the cDNA encoding the del locus of Antirrhinum majus has been cloned is described in more detail below. By making use of the cDNA sequence set out in SEQ ID NO 1, a DNA molecule encoding the DEL protein can now be obtained as required using standard techniques of cDNA cloning and/or DNA synthesis. DNA molecules encoding a protein having an amino acid sequence which shows WO 93/14211 PCT/GB93/00019 homology with the DEL protein as shown in SEQ ID NO 1 or 2 can be obtained by mutation of a DNA molecule having the sequence shown in SEQ ID NO 1 using standard techniques of recombinant DNA technology and/or by DNA synthesis.

Utilizing standard techniques the DNA molecule encoding a protein having the amino acid sequence of the DEL protein as shown in SEO ID NO's 1 or 2 or a protein having an amino acid sequence which is at least 80%, preferably at least more preferably at least 98% similar with the DEL protein as shown in SEQ ID NO 1 or 2 or protein encoded thereby may also be used to isolate DNA molecules encoding a protein having an amino acid sequence which shows homology with the DEL protein as shown in SEQ ID NO 1 or 2 from other plant species, most preferably plant species belonging to the dicotyledoneae.

For use according to the present invention the DNA segment encoding the DEL protein or a protein homologous thereto will generally be incorporated in an expression vector which also includes suitable regulatory and control sequences to enable expression of the segment in a particular plant or part of a plant. Examples of suitable promoters include the cauliflower mosaic virus 35S promoter and also any promoter which is expressed in epidermal cells of different plant organs, such as the promoter of a housekeeping gene or a gene for the synthesis of specific epidermal structures. By use of a promoter which is specific for a particular type of plant tissue, i.e. is effective only in that tissue, the effect of the DEL protein or the protein homologous thereto can be confined to that specific tissue.

Plants transformed with the DNA segment encoding the DEL protein or a protein homologous thereto may be produced by standard techniques which are already known for the genetic manipulation of plants. For example the DNA segment may be incorporated into an Agrobacterium vector and plant material may then be infected by a strain of Agrobacterium carrying this vector. In this way the DNA ,WO 93/14211 PCT/G B93/00019 encoding the DEL protein or a protein homologous thereto becomes integrated into the genome of the plant tissue so that plants propagated from the tissue also carry this DNA.

Alternative methods for the introduction to the DNA into plant cells include precipitation onto tungsten particles and shooting using a particle gun.

The ability to mediate or control the expression of anthocyanin pigmentation in plants can be put to practical use in a number of ways. Thus plant pigmentation can be increased or altered by transforming plants in the manner described above with a construct including the DNA encoding the DEL protein or a protein homologous thereto under control of a suitable promoter. By use of a construct in which a regulatory sequence, such as the promoter, is specific to a particular part of the plant, for example specific parts of the flower, the effect on pigmentation can be confined to that part of the plant.

In particular, the procedure described above can be used to enhance pigmentation of regions already pigmented in the host species or to pigment areas which are not normally pigmented in the host species. One specific application of this procedure is to produce novel genetically manipulated flowering plants with a phenotype in which the flowers show a pattern or intensity of pigmentation which differs from the host species.

It is possible to use the DNA segment encoding the DEL protein or a protein homologous thereto as a promoter/enhancer trap wherein the DNA segment which is driven by a "minimal" promoter, i.e. a truncated promoter more or less deficient in cis-acting regulatory sequences, may be cloned within a transposon (which can only transpose when a second gene containing the trans-activator is also present) and the construct is then introduced into plants.

The transposon containing the DNA segment can then transpose to a new site near to a variety of promoters/ enhancers which can increase or activate transcription of the DNA segment. It is thus possible to derive and select PCT/B 3 0 0 0 19 14 FEBRUARY 1994 new pigmentation patterns by simply screening progeny from the transgenic plants. The transposon can subsequently be "stabilised" by crossing out the factor that activates the transposon. The chimeric sequence combining the DNA segment with the trapped promoter/enhancer can be recovered by reisolating back the construct, together with adjacent sequences, from the plant and used to control the expression of any gene of interest.

In particular the trapped promoter/enhancer may be used to control expression of a gene of interest in two ways, namely: 1) A new construct is prepared which comprises a gene of interest under the control of an anthocyanin gene promoter upstream and operably linked thereto. When this new construct is transformed into a plant, wherein a promoter/enhancer has been trapped, expression is seen of the gene of interest in those cells which express delila.

The trapped promoter/enhancer causes expression of the DEL protein which in turn switches on the anthocyanin promoter thus causing expression of the gene of interest.

2) In the re-isolated construct the DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which is at least preferably at least 90%, more preferably at least 98% homologous with the DEL protein as shown in SEQ ID NO 1 or 2 is excised from the construct and a heterologous gene of interest inserted in its place to form a new construct.

When this new construct is transformed into a plant the heterologous gene of interest is expressed.

It may also be desirable to reduce plant pigmentation either in localised areas or throughout a plant. This may be achieved by various techniques such as those, for example, based on the use of DNA sequences showing sequence homology to del including antisense RNA, co-suppression or ribozymes. These techniques may be defined as follows: Antisense RNA is where a gene is expressed in the opposite sense to normal the promoter is at the 3' end of the T E. S 21 r 0 4 1^^ .^TTT HE .WO 93/14211 PCT/GB93/00019 gene), such that the "wrong" strand of the DNA is transcribed into RNA (giving antisense RNA). This antisense RNA may form a duplex with normal sense RNA and so inactivate it.

Co-suppression occurs where extra copies of a gene are introduced into the genome which may result in inactivation of the endogenous gene, which may in turn cause a mutant phenotype.

A Ribozyme is an RNA molecule which has the property that when it hybridizes to another RNA molecule containing a particular nucleotide sequence (target sequence), it will cleave the molecule and hence inactivate it. The nature of the target sequence depends on the critical region in the ribozyme molecule which is complementary to the target.

The particular RNA molecule that the ribozyme recognises can therefore be altered by changing the critical region of the ribozyme.

In addition, constructs can be developed with alterations in the del coding sequence which produce proteins which interfere with the functioning of delila or delila-like genes in the host species.

The del coding sequence or a homologue thereof can also be used as a convenient visible marker. Thus use of a DNA sequence encoding the DEL protein or a protein homologous thereto in the manner described above allows the coding sequence in question to act as a visible marker for gene expression. This can be exploited to enable easy identification of transformed cells, cells in which a particular promoter is active, cells in which gene functions have been activated or inhibited, e.g. by excision or integration of a transposon. A DNA segment encoding the DEL protein or a protein homologous thereto in the manner described above may be used as a visible marker in a transgenic plant line, most particularly including dicotyledonous species, wherein a specific pigmentation pattern may be used to identify the line.

WO 93/14211 PC1'/G B93/000 19 The del coding sequence or a homologue thereof can also be used to trans-activate or inhibit genes by placing the gene of interest under the control of a promoter, e.g.

the pallida promoter of Antirrhinum, already known to be regulated by the delila gene. Plants containing such constructs, together with the del coding sequence (or an appropriate homologue thereof), would express the gene of interest only in those cells which express delila. This could be combined with the use of the del coding sequence as a visible marker so that cells expressing the gene of interest could be identified by their pigmentation phenotype.

A further possible use for the del coding sequence is in the isolation of homologous of the delila gene from various plant species. Such homologous of the delila gene can be isolated using genomic or cDNA probes derived from delila clones or based on the del coding sequence as set out in SEQ ID NO 1.

The invention is based on and further illustrated by the following experimental work in which reference is made to the accompanying drawings. It is to be noted that the experimental work utilizes del, however, clearly other DNA segments which encode a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which shows substantial homology with the DEL protein as shown in SEQ ID NO 1 or 2 and which is capable of regulating expression of one or more plant genes involved in pigment biosynthesis. A brief description of each figure of the accompanying drawings is as follows.

FIGURE 1 nucleotide and predicted amino acid sequence of del cDNA.

FIGURE 2 Southern blots of EcoRl digested genomic DNA from various Antirrhinum majus plants.

FIGURE 3 Sequence comparisons of Del+ and del-602 alleles in the region of the Tam2 insertion.

FIGURE 4 Northern analysis of del expression in WO 93/14211 PCT/G 193/00019 various Antirrhinum majus flowers at different stages of development.

FIGURE 5 Amino acid sequence comparison of DEL protein with selected HLH proteins.

FIGURE 6 In situ hybridisation of medial longitudinal sections of corollas with 35S labelled RNA probt:.

7 Plasmids pBJIMM15, pBJIMM21 and pBJIMM24.

1. Isolation of th! del gene A large scale transposon mutagenesis experiment yielded various homeotic and pigmentation mutants (Carpenter and Coen, Genes Dev., 4, 1483-1493, (1990), Luo et al., The Plant Journal, 1, 59-69 (1991). One mutation gave a phenotype similar to that of the existing del mutation, and was shown to be a recessive del allele, del- 602 (Luo et al., ibid). Self pollinated del-602 plants gave about 7.5% of progeny with a wild-type phenotype (revertants), explicable if a transposon excision occurred in germinal tissues of the parent plants. To identify the transposon at the del locus, genomic DNA from del-602 mutant and revertant plants was digested with restriction enzymes and probed with the various transposable elements isolated from A. nrajus. Because each of these elements was present in multiple copies in the genome, several bands were seen in Southern blots. When EcoRI digested DNA was probed with a fragment of the transposon Tam2 (Upadhaya et al., Mol. Gen. Genet., 199, 201-207 (1985), a 5.6 kb band was consistently observed in mutants and not in revertants (Figure 2a). This suggested that the del-602 mutation resulted from a Tam2 insertion, and the 5.6 kb fragment was therefore cloned.

The resulting clone, pJAM 602, contained a 4.9 kb fragment of Tam2 with 0.7 kb of flanking DNA (Figure 2b).

The flanking sequence (probe A) was then used to probe EcoRI digested DNA from various genotypes: del-602 plants showed the expected 5.6 kb band; plants of the progenitor PCT/GB 9 3 00 1 9 14 FEBRUARY 194 showed the expected 5.6 kb band; plants of the progenitor stock showed a wild-type band of 6.2 kb; and plants homozygous for a stable del allele, del-8, gave a 2.8 kb band (Figure 2b). If probe A derived from the del locus, reversion of the del-602 allele to wild-type should have correlated with restoration of the wild-type 6.2 kb band, and this was observed; six revertant plants obtained in the progeny of three crosses between del-602 and del-8 plants, and therefore representing at least three independent germinal reversions of the del-602 allele, all showed the 6.2 kb band, confirming that the pJAM 602 clone contained part of the del locus.

A clone of the Del+ genomic region was obtained by screening a genomic library from the progenitor stock with probe A of pJAM 602 (Figure 2b). A comparison of the sequences flanking the Tam2 insertion in the del-602 allele with the corresponding wild-type sequences identified a direct duplication of 3 base pairs of target DNA, a length characteristic of Tam2 insertions (Upadhaya et al, ibid) (Figure 3).

Figure 2(a) shows Southern blot of EcoRI-digested genomic DNA from del-602 mutant and revertant (Del plants, probed with a 4.4 kb EcoRI/HindIII fragment of the Tam2 clone pRH2, provided by Enno Krebbers and Hans Sommer.

These plants were obtained in the F 1 progeny of a cross between del-602 and del-8 plants. Revertants have the presumed genotype Del/+del-8, and mutants del-602/del-8.

A restriction map of Tam2 and the origin of the probe are shown below the autoradiograph. Sites indicated are EcoRI HindIII and BglII Figure 2(b) shows Southern blot of EcoRI-digested genomic DNAs probed with fragment A of the 5.6 kb EcoRI clone, pJAM 602. Lane 1, wild-type progenitor of del-602; lane 2, homozygous del-602; lane 3, homozygous del-8; lanes 4-9, revertant progeny from crosses between del-602 and del-8 plants; lanes 10-16 del mutant progeny from the same crosses. A restriction map of the 5.6 kb EcoRI clone, pJAM 13 WO 93/141211 PCT/GB93/00019 line, Tam2 sequences; thin line, flanking sequences.

RNA extraction and Southern blot analysis were performed as described by Coen et al., Cell 47, 285-296 (1986). The pJAM 602 clone was obtained by digesting genomic DNA of del-602 plants with EcoRI, gel-purifying fragments in the 5-6 kb size range, ligating to ANM1149 arms and screening a library of 30,000 plaques with the Tam2 probe shown in Figure 2a. The resulting clone was subcloned into Bluescript SK (Stratagene).

Figure 3 shows a sequence comparison of Del and del- 602 alleles in the region of the Tam2 insertion. The target sequence, duplicated on insertion of Tam2, is boxed.

The pJAM 602 clone was sequenced to provide flanking sequences to the right of the Tam2 insertion. To obtain the flanking sequence to the left of Tam2, 0.1 gg of genomic DNA from a del-602 plant was amplified by PCR (Saiki et al, Science, 239, 487-491 (1988), using a primer derived from sequences near the left terminus of Tam 2 (as orientated in Figure 2b) and a second primer based on Del genomic sequences. The expected fragment of 0.3 kb was subcloned to give pJAM 122 and sequenced.

2. Characteristics of the putative DEL protein and expression of del To identify the Del transcript, polyA RNA extracted from corolla tubes was hybridised with probe A of pJAM 602.

A single 2.5 kb transcript, detected in wild type tubes, was absent from the flowers of both del-602 and del-8 mutants (Figure 4a). A cDNA library prepared from wild type flower buds was screened with probe A and several cDNA clones were isolated and sequenced. The cDNA sequence (SEQ ID NO 1) contained a long open reading frame (ORF) starting at position +25 with an ATG codon flanked by sequences which conformed to the consensus for initiation of translation in plants (LUttke et al., EMBO 6, 43-48 (1987). The ORF encoded a potential protein, DEL, of 644 amino acids. Comparison of the amino acid sequence of DEL to proteins on the PIR and SWISS databases using the FASTA WO 93/14211 PCT/GB93/00019 program (Lipman and Pearson, Science, 227, 1435-1441 (1985) revealed a strong homology betwnen DEL and the products of Lc (Ludwig et al., ibid) and R-S (Perrot and Cone, Nucl.

Acids Res., 17, 8003 (1989)), two members of the R gene family which control the pigmentation pattern in maize.

The deduced amino acid sequences of the products of del, R and Lc had 38% identity when optimally aligned (58% similarity, allowing for conservative changes). Two regions of DEL were highly conserved; near the N-terminus, residues 16-190 were 61% identical with the corresponding regions of R-S and Lc proteins, and towards the carbo' terminus, residues 438-497 were 60% identical. The latti region also resembled a helix-loop-helix (HLH) domain conserved in a number of eukaryotic regulatory genes (SEQ ID NO's 3 to 12). The strongest similarity was to myc proteins, involved in animal cell proliferation control, and the human transcription factor E3, which binds to the immunoglobin heavy chain enhancer motif AE3.

DEL also contained a highly acidic region (residues 173-319); 27 acidic and two basic residues gave an overall negative charge of -25. A corresponding acidic region occurs in Lc (Ludwig et al., ibid), but relatively little conservation in amino acid sequence was found between these regions of Lc and DEL (25% identity). The conserved region near the N-terminus of DEL showed no significant homology to proteins other than those of the R gene family.

Secondary structure analysis (Chou and Fasman, Biochemistry, 13, 211-244 (1974)) of this region identified several sequences predicted to form a helices, one of which (residues 79-89) appeared strongly amphipathic in helical wheel plots and was highly conserved between DEL and Lc.

Alignment of the 'sequence of the del-602 genomic clone with the cDNA sequence showed that Tam2 was inserted in an intron, 285 nucleotides upstream of the 3' intron-exon junction (Figure 1 and SEQ ID NO Northern analysis of RNA from del-602 flowers revealed various aberrant-sized transcripts (Figure 4a), which could result from the Tam2 NWO 93/14211 PCI'/G B93/00019 insertion interfering .'ith processing of the primary del transcript.

A del cDNA clone was used to screen a wild-type genomic library and three clones with extensive homology to del were isolated. Detailed restriction mapping indicated that they derived from independent loci, distinct from del.

Therefore, A majus has a family of at least four genes related to del.

To determine the temporal pattern of del expression, a Northern blot of RNA from different-sized flower buds was probed with a del cDNA (Figure 4b). A low level of expression was detected in the youngest flower buds examined and was maintained until shortly before anthesis (correlated with flower opening) when expression increased and then declined. The rise in del expression occurred at about the same time that flowers became strongly pigmented.

However, there was detectable expression considerably before flower buds were visibly pigmented. This could imply that a threshold level of del product was necessary for anthocyanin biosynthesis, or that additional factors were required at these early stages for del to activate its target genes. To compare the expression of del with one of its target genes, the Northern blot was stripped and reprobed with a cDNA clone of pallida (pal), a biosynthetic gene strongly regulated by del (Figure 4b. Almeida et al, ibid). At the later stages of flower development the pattern of pal expression closely resembled that of del, but the earliest detectable pal expression was after that of del, implying that del expression was not sufficient to activate pal at very early stages. The spatial pattern of del expression in the corolla was determined by Northern analysis of RNA from dissected wild-type corollas (Figure 4c). Pigmentation of the corolla first appeared in the lobes and in a ring at the base of the tubes, and subsequently extended throughout the tubes. The strongest expression of del was seen at the base of the tubes, the region of pigmentation most greatly affected by the del WO 93/14211l PCT/G 93/00019 mutation. Expression was also detected in the lobes, as was predicted from genetic interactions described previously (Almeida et al., ibid). As expected, no wildtype del transcript was found in the pigmented lobes of del mutant flowers (results not shown), confirming that del expression is not required for pigment biosynthesis in the lobes. Probing of a Northern blot of RNA from diverse organs with del cDNA showed the strongest expression in the corolla, stamens and style, with a weak signal in sepals and carpels, and little or none in bracts, leaves and stem (Figure 4d). All organs in which del expression was detected were visibly pigmented, and the level of del expression correlated with the degree of pigmentation.

The expression of del was further localised by in situ hybridisation of 35S-labelled del RNA to sections of wildtype corollas. Signal was detected only when the antisense strand of del was used as a probe, and was strongest in the flower buds 1-6 nodes above the first fully opened flower on the inflorescence. The signal was specific to the epidermal cell layers in both tubes and lobes (Figure 6).

This corresponds to the distribution of anthocyanins, and of expression of the biosynthetic genes nivea, pallida and incolorata (Jackson, Current Biology, 99 (1991)). The two epidermal layers of the petal are referred to as inner (lining the throat of the tube and contiguous surface of the lobes) and outer (lining the exterior surface of the tube and lobes). In the corolla tubes, signal was as intense in the outer epidermis as in the inner epidermis (Figure 6a). The outer epidermis of the tubes and lobes bore numerous multi-cellular hairs, which were unpigmented; no signal was seen in these hairs (data not shown). In the corolla lobes, a strong signal was also seen in both the outer and inner epidermi. However, in sections from the central face of the flower, the signal was most abundant in the inner epidermis (Figure 6b), which also had strongest pigmentation.

Figure 4 shows Northern analysis of del expression.

,WO 93/14211 PCT/GB93/I00 9 Northern blot of RNA from the corolla tubes of DEL del-8 and del-602 flowers, hybridised with probe A of pJAM 602 (Figure 2b). Each lane contained 34g of polyA+RNA.

Note the hybridizing band at 2.5 kb. RNA from flower buds of different sizes, as indicated by diagrams above each track. Nodes on the Antirrhinum inflorescence (a raceme) bear flower buds at progressively older developmental stages in a series from apex to base. RNA was extracted from flowers at different positions on the inflorescence; 21-28 nodes above the first fully opened flower (lane 17-20 nodes above (lane 13-16 nodes above (lane 9-12 nodes above (lane 5-8 nodes above (lane 1-4 nodes above (lane and the first four fully opened flowers (lane 7).

The Northern blot was first probed with the del cDNA clone pJAM 121, then after autoradiography it was stripped and reprobed with a pallida (pal) cDNA clone pJAM 225, provided by C. Martin. The pal gene encodes the enzyme dihydroflavonol reductase involved in anthocyanin biosynthesis, and is known to be regulated by del (Almeida et al, Genes Dev., 3, 1758-1767 (1989)). RNA from wild-type flower buds (1-5 nodes above first fully open flower) dissected into three parts: the base of corolla tubes the rest of the tube and the lobes as indicated above the autoradiogram, probed with the del cDNA clone pJAM 121. RNA from leaves stems bracts sepals petals stamens (St) styles (Sty) and carpels probed with the del cDNA clone pJAM 121.

Each lane of and contains 10pg of RNA, and loading appeared equal when ribosomal bands were viewed under UV illumination after staining with ethidium bromide.

RNA extraction and Northern analysis was carried out as described (in Coen et al., ibid). Northern blots were stripped by washing in 0.5% SDS, 0.01 SSC for 30 minutes at 80 0

C.

Nucleotide and predicted amino acid sequence of del cDNAs is shown in Figure 1 and SEQ ID NO 1. The predicted 'WO 93/14211 PCIT/GB93/00019 amino acid sequence of del is also shown in SEQ ID NO 2.

The solid triangle indicates the position of an intron within which Tam2 is inserted in the del-602 allele, namely between bases 573 and 574. The region of the DEL protein with similarity to the helix-loop-helix domain of the myc family of transcription factors is underlined, it commences at residue 439 and terminates at residue 493. Residues conserved in DEL and the maize R-S gene product (after alignment) are shaded.

Figure 5 and SEQ ID NO's 3 to 12 show amino acid sequence comparison of DEL protein with selected HLH proteins. Alignments were made to maximise homology within the HLH domain. A consensus sequence derived for residues conserved in most known HLH genes is shown below (Benezra et al., Cell, 61, 49-59 (1990) and Cai and Davis, Cell 61 437-446 (1990)) Shaded regions identify residues that match the consensus. The positions of the conserved basic region, putative amphipathic helices I and II, and the loop are shown above (Murre et al, Cell, 56, 577-783 (1989)). The sequences shown are for: maize R-S (Perrot and Cone, ibid) and Lc (Ludwig et al., ibid); human E3 (Beckman et al, Genes Dev., 4, 167-179 (1990)), L-myc (DePinho et al, Genes Dev., 1, 1311-1326 (1987) and N-myc (Kohl et al, Nature, 319, 73-77 (1986)); yeast Cbfl (Cai and Davis, ibid); AP4 (Hu et al, Genes Dev, 4, 1741-1752, (1990)); B (Radicella et al, Plant Mol. Bil., 17, 127-130 (1991)) and mouse myogenin (Edmondsen and Olson, Genes Dev., 3, 628-640 (1989)).

cDNA was synthesised from 34gpolyA+RNA extracted from wild-type flower buds (1-4 nodes above first fully opened flower), and cloned into the EcoRI site of ANM1149 using Amersham kits. A library of l05 plaques was screened with probe A of pJAM 602 (Figure 2b) and the longest clone obtained was subcloned into Bluescript SK (Stratagene).

Sequence analysis revealed that the cDNA insert lacked a poly A tail but contained a long open reading frame which terminated at an EcoRI site, suggesting that the cDNA had 'WO 93/14211 PCT/GB93/00019 been cleaved at an internal site during the cloning.

Alignment of genomic sequences of pJAM 602 with the cDNA showed that the probe terminated at the corresponding EcoRI site, so the library was rescreened with a fragment of a genomic Del+ clone extending 3' of the original probe. A further clone was obtained and contained an insert with sequences extending from the EcoRI site to the poly A tail.

To c.nfirm that the two sequences were contiguous, an intact clone, pJAM 121, was isolated by PCR amplification of cDNA ends RACE) (Frohman et al., Proc. Natl. Acad.

Sci. 85, 8998-9002 (1988)) using a specific primer based on sequences from the 5' end of the cDNA sequence, and this was sequenced around the EcoRI site. The del cDNA sequence is about 0.4 kb smaller than the observed transcript, suggesting that it is not full length.

However, alignment of DEL with Lc/R proteins suggests that the cDNA sequence contains the entire del coding sequence.

Sequences were determined by the plasmid dideoxynucleotide sequencing method (Chen and Seeburg, DNA 4, 165-170 (1985)) using a Sequenase kit (USB), and both strands of cDNAs were sequenced. Computer analysis of sequences was performed using the University of Wisconsin Computer Group programs.

Figure 6 shows in situ hybridisation of medial longitudinal sections of corollas with 35 S labelled RNA probes. Tube tissue under light (left) and dark field (right). The inner epidermis mesophyll and outer epidermis are labelled. Silver grains corresponding to del expression are seen in dark field, and are localised to the epidermis in inner and outer surfaces of the tube. (b) Lobe tissues viewed under light field (left) and dark field (right). Silver grains are at high density over the inner epidormis, and at a lesser density over the outer epidermis.

Corollas were fixed in 4% paraformaldehyde and embedded in wax, and sections were prepared for hybridisation as described by Jackson, Molecular Plant Pathology: A Practical Approach (Bowles, Gurr, McPherson WO 93/14211 PCT/GB93/00019 eds) Oxford University Press (1991). A 1kb fragment of the del cDNA encoding the N-termimus but not the HLH region of DEL was subcloned into Bluescript SK and KS These plasmids were linearised with restriction enzymes cutting in the polylinker furthest from the T7 promoter. 35 S RNA probes were synthesised using 1pg of linearised plasmid as template, t7 polymerase and 50 pCi of 35 S UTP (1300 mCi/mmol, NEN). Transcription reactions, hybridisation, and autoradiography were carried out as described by (Ingham et al, Nature, 318 439-445 (1985)).

Autoradiographic exposure was for 14 days at 4 0 C. No signal above background was observed using control sensestrand probes.

3. Transformation of both tomato and tobacco with the del gene A number of independent transformed lines, of both tomato and tobacco, have been produced using three molecular constructs that utilise the del gene. The transformation of both tomato and tobacco was performed through a standard procedure utilising Agrobacterium tumefaciens as detailed below.

Tomato: Approximately 100 tomato seed (Lycopersicon esculentum variety Money Maker) were surface sterilised using a 10% aqueous solution of bleach and sown on agar media under sterile conditions (day The seeds were allowed to germinate and grow for 10 days (day On day 9 a 10ml volume of luria broth was inoculated with Agrobacterium tumefaciens strain Ach5 carrying the plasmid pAL4404 (Hoekema et al, Nature, 303, 179-180 (1983)) and one of the following plasmids, pBJIMM21 or pBJIMM24. The plasmids can be seen in Figure 7 and are derived from the plasmid pSLJ456 (derived by J.

D. Jones C. Dean et al from pRK290 J. D. Jones C. Dean et al 77, 7347, (1980)). As can be seen in the diagrams pBJIMM15 carries the cDNA of the del gene of Antirrhinum majus under control of the cauliflower mosaic virus 35S promoter and is terminated by the OCS gene 3' WO 93/1421i PCf/GB93/00019 terminator sequence, pBJIMM21 carries a copy of the del gene derived from a genomic fragment again driven by the promoter, and pBJIMM24 carries a large genomic fragment containing both the promoter and coding sequence of del.

These three plasmids all also carry the NPT gene that confers kanamycin resistance and this gene is driven by the 2'1' promoter (Velten et al. EMBO 3, 2723-2730, (1984)) again terminated by the OCS 3' sequence. The three different cultures (differing only in the type of pBJIMM plasmid that they contained) were allowed to grow for two days (until day 11). The transformation procedure employed was identical for each type of culture and therefore will only be detailed once.

On day 11 the tomato seedlings were cut into small pieces to produce tissue explants. These explants were washed with the culture of Agrobacterium tumefaciens described above and the bacterium was allowed to remain in contact with the explants for 2 days (to day 13).

On day 13 the tissue explants were placed on selective agar plates bearing appropriate antibiotics to kill any remaining Agrobacterium and allow only "transformed" plant tissue to regenerate. The period between days 11 and 13 when the Agrobacterium is co-cultivated with the tomato tissue is to allow time for the Agrobacterium to transfer the portion of DNA from the pBJIMM plasmids that lie between the points marked "left border" and "right border" in Figure 7 into the genome of the tomato. Once incorporated into the plant genome, the NPT gene becomes functional confers kanamycin resistance to the tomato tissue and thereby allowing it to regenerate on agar medium containing kanamycin.

Over the next two to six weeks (days 27 to 55) calli and shoots regenerated from the explants. Small shoots were removed and transplanted into media containing kanamycin. Only shoots that derive from "transformed" tissue will produce roots on this media. For all pBJIMM constructs rooting was observed with on average 10% of WO 93/14211 PCT/GB93/00019 shoots transferred to Kanamycin containing rooting media.

Rooting was found to occur over one to three weeks (day 62 to 76). When rooted shoots were approximately 5cm tall they were transferred to peat based potting compost and grown on in the glasshouses for 6 weeks (to day 118).

Through this procedure 10 plants were regenerated from the pBJIMM15 transformation seven of which have shown enhanced anthocyanin pigmentation phenotypes. 9 Plants from the pBJIMM21 transformation seven of which show enhanced pigmentation and one plant from the pBJIMM24 transformation that shows a small increase in pigmentation were obtained. Southern blot hybridisation analysis revealed that all plants -howing pigmentation had incorporated the del construct from between the left and right border points of the pBJIMM plasmid. The phenotypes of the plants from the pBJIMM15 and pBJIMM21 transformations were very similar. The shoots and leaves were much more heavily pigmented with anthocyanin than the control plants. When flowers were produced they were observed to have a stripe of purple pigmentation visible over the main vein of the petal, a phenotype absence from the control. When the roots of these plants were exposed to light it was found that purple anthocyanin pigment was accumulated, in contrast to a very low level of pigmentation observed in controls. When these tissues were sectioned it was found that the pigment was being produced in the epidermal and sub-epidermal layers of the leaf, the epidermal and sub-epidermal layers of the stem and only in the sub-epidermal layer of the root.

Tobacco: The tobacco transformation procedure is very similar to that of tomato. The same cultures of Agrobacterium were used, however, plants have only been regenerated from pBJIMM15 and pBJIMM21 cultures. On day 0 ml volumes of Luria broth were inoculated with Agrobacteriu cultures as previously described, in this case only cultures bearing pBJIMM15 and 24 have been used.

On day 2 several immature leaves of tobacco (Nicotiana WO 931/14211 PCT/GB93/0001 9 tabaccum variety Samson) were harvested from plants growing in the greenhouse. These were then surface sterilized by washing in a 10% solution of bleach for five minutes and then rinsed in sterile water and cut into small tissue explants. The explants were then washed with the Agrobacterium cultures and allowed to co-cultivate for two days (days 3 to 4).

The explants were then transferred to agar plates containing kanamycin and over the next 2 to 3 weeks tissue regeneration occurred resulting in the production of small shoots (days 15 to 21). These were excised and rooted in media containing kanamycin, after producing roots and reaching a height of approximately 5cm these plantlets were transferred to a peat based potting compost and grown on in the greenhouse to maturity in approximately 2 months.

Through this method 7 plants have been produced from the culture and of these 5 show enhanced pigmentation, 9 plants have been produced from the pBJIMM24 transformation and 4 show a slight enhancement of pigmentation. By southern analysis it was shown that plants which exhibited a change in pigmentation also carried the del construct from the pBJIMM plasmid. The pigment phenotype of the transformed tobacco was visible only in the flowers of the plant. The flower petals and anther filaments of the transformants were much more intensely pigmented than similarly cultivated control plants. On sectioning this pigmentation appeared to be epidermal in the flower and both epidermal and subepidermal in the anther filament. The phenotype was only observed in the flower as the shoots, stem and leaves of the transformants were all indistinguishable from the control plant.

WO 93/14211 PCT/GB93/00019 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: COEN, Enrico STREET: John Innes Institute, Colney Lane CITY: Norwich STATE: Norfolk COUNTRY: Great Britain POSTAL CODE (ZIP): NR4 7UH TELEPHONE: (0603) 52571 TELEFAX: (0603) 56844 TELEX: 975122 (JIINOR G) NAME: CARPENTER, Rosemary STREET: John Innes Institute, Colney Lane CITY: Norwich STATE: Norfolk COUNTRY: Great Britain POSTAL CODE (ZIP): NR4 7UH TELEPHONE: (0603) 52571 TELEFAX: (0603) 56844 TELEX: 957122 (JIINOR G) NAME: GOODRICH, Justin STREET: John Innes Institute, Colney Lane CITY: Norwich STATE: Norfolk COUNTRY: Great Britain POSTAL CODE (ZIP): NR4 7UH TELEPHONE: (0603) 52571 TELEFAX: (0603) 56844 TELEX: 957122 (JIINOR G) NAME: FREEDMAN, Roger STREET: Diatech Limited, 19 Millers Yard, 10/11 Mill Lane, CITY: Cambridge COUNTRY: Great Britain POSTAL CODE (ZIP): CB2 IRQ TELEPHONE: (0223) 69936 TELEFAX: (0223) 464113 TITLE OF INVENTION: Regulation of Plant Genes (iii) NUMBER OF SEQUENCES: 12 (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) CURRENT APPLICATION DATA: .WO 93/14211 PCT/GB93/00019 APPLICATION NUMBER: PCT/GB93/ (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 07/818,570 FILING DATE: 09-JAN-1992 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 2075 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE

ORGANISM:

(ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

Antirrhinum Majus

CDS

25..1956 insertion_seq (573"57 4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GTAGAGAGGA GAGAGGATTC AAGA ATG GCT ACT GGT ATC CAA AAC CAA AAG Met Ala Thr Gly lie Gin Asn Gin Lys GTC CCT GAG Val Pro Glu AAT TTC AGG AAG CAA CTT GCT ATT GCT GTG AGA ACT Asn Leu Arg Lys Gin Leu Ala lie Ala Val Arg Ser 20 ATC CAA TGG AGT lie Gin Trp Sar GCA ATT TTC TGG Ala Ile Phe Trp AAT TCA GTT GCA CAA CCA Asn Ser Val Ala Gin Pro GGG GTC TTG Gly Val Leu CGA AAA ACT Arg Lys Thr TGCG GGT GAT CCCGG TTC TAC AAT GGA Trp Gly Asp Gly Phe Tyr Asn Gly GAT ATT AAA Asp Ile Lys GTA CAA TCT GTC CAA TTG AAT CAA GAT CAG CTC GGA TTG Val Gin Ser Val Clu Leu Asn Gin Asp CGn Leu Gly Leu CAG AGA Gin Arg ACT GAT CAA TTG Ser Asp Gin Leu GAA CTT TAT GAG Clu Leu Tyr Glu CTT TCA CTT GCT Leu Ser Leu Gly ,WO 93/14211 PCT/GB3/00019 ACC AAC ACA CM-A Thr Asn Thr Gin AAA AGG CCT ACT Lys Arg Pro Thr GCA TTA TCA CCA Ala Leu Ser Pro GAC CTC ACT GAT Asp Leu Thr Asp GAG TGG TTT TTC TTG GTT TGC ATG TCT Giu Trp Phe Phe Leu Val Cys Met Ser 115 TTC ATA Phe Ile 120 TTC AAT ATT Phe Asn Ile GCA GTA TGG Ala Vai Trp 140 CAA GGG TTG CCT Gin Gly Leu Pro AGA ACA TTA GCA Arg Thr Leu Ala CGA MAT CAA Arg Asn Gin 135 GTT TTC TCG Val Phe Ser CTA TGC AAC GCT Leu Cys Asn Ala CGT GCG CAC ACC Arg Ala Asp Thr CGT TCT Arg Ser 155 TTG CTT GCA AAG Leu Leu Ala Lys

AGT

Ser 160 GCG TCA ATT CAG Ala Ser Ile Gin GTT GTG TGC TTT Val Vai Cys Phe

CCA

Pro 170 TAT TCA CAA GGT Tyr Ser Giu Gly GTT GAG CTG GGA Val Giu Leu Gly ACA GAG CTA GTA Thr Giu Leu Val GAG GAT TTG AAT Glu Asp Leu Asn ATC GAG CAT ATA Ile Gin His Ile ACT TCA TTC TTG Thr Ser Phe Leu GAG AGT Asp Ser 200 CCT GCC ACC Pro Ala Thr MAC AAC AAT Asn Asn Asn 220 CCC AAG ATT CCC Pro Lys Ile Pro TAT GTC TCC AAC Tyr Val Ser Asn AGT ATT ACA Ser Ile Thr 215 MAT ATA CCA Asn Ile Pro CAC CTC ATT TGT Asp Leu Ile Cys GCG CTT GAA CAT Ala Leu Giu His GAA AAC Glu Asn 235 GAT CTT CAT GAG Aip Leu Asp Gin TTG M-T TGT CCA Leu Asr Cys Pro ACG AAC ATA TGT Thr Asn Ile Gys

TCT

Ser 250 CCT GAT AAC ACT Pro Asp Asn Ser GAT GAG TTT GCA Asp Asp Phe Ala

GAC

Asp 260 AAT TTA CTC ATA Asn Leu Leu Ile GAA TCC AAT TTG Glu Ser Asn Leu

GCA

Ala 270 GAA GGC ATC AT Glu Gly Ile Asn GAG GTT CCT CAA Giu Val Pro Gin ACA CAA Thr Gln 280 AGC TGG CCT Ser Trp Pro ATG AAT TCT Met Asn Ser 300 ATG GAT GAT GCA Met Asp Asp Aia ACC AAT TGT CTC Ser Asn Cys Leu AAT AGT TCT Asn Ser Ser 295 CTA GAG TCT Leu G~u Ser ACT CAC TGT ATA Ser Asp Cys Ile CAA ACT CAT GAA Cln Thr His Glu WO 93/14211 WO 9314211PCI/C B93/OOO 19 TTT CCT CCA CTT TCT CAT Phe Ala Pro Len Ser Asp A.AA OGG CCA CCC Lys Cly Pro Pro ACC AAT AAT TCT Thr Asn Asn Cys CAC AGC ACT CAA His Ser Thr Cmn TCC AAT GAG GAG Cys Asn Cln Gin CAA MAC ACC GGT Gu Asn Thr Gly 1011 1059 1107 1155 CAA CCC CAT GAG Gin Gly Asp Gin CAT TAT CAA CCC His Tyr Gin Gly CTT TCC MAT CTT Leu Ser Asn Leu TTG MAG Leu Lys 360 ACT TCC CAT Ser Ser His CAA TCA ACC Gin Ser Ser 380 TTC GTT CTT CCT CCC TAG TTC AGA MAT Leu Val Len Gly Pro Tyr Phe Arg Asn 370 CGG MAT AGA Gly Asn Arg 375 GCT ACT CAT Gly Thr His TTC GTT ACT TCC Phe Val Set Trp MAC CAT CCA TCC Lys Asp Cly Ser 1203 CTT CCC Val Pro 395 CGA AGC CCA ACC Arg Set Cly Thr CAA ACA TTT CTC Gin Arg Phe Len MAA CTA CTT TTT Lys Val Leu Phe CTA CCT AGA ATG Val Aia Arg Met CMA MC TCC ACC Gin Asn Ser Arg CAT CCT CCT AMA Asp Ala Cly Lys

GMA

Gin 425 1251 1299 1347 MCG CCC MAC ACT Lys Cly Asn Ser TCC CTT GCA MCG Cys Len Ala Lys ACC GGT CAT GMA Thr Ala Asp Gin ATT CAT Ile Asp 440 AGA MAC CAC Arg Asn His CCC TTT ATC Arg Phe Met 460 TTC TCA GAG AGA Len Set Gin Arg CCC AGA GAG A Arg Arg Giu Lys ATA MAC CMA Ile Asn Gin 455 MCG CTT GAC Lys Vai Asp 1395 1443 ATT CTT GCA TCC lie Leu Ala Ser CTC CCA TCCGGCT Val Pro Ser Ciy AA CTA Lys Val 475 TCA ATA CTA GAC Se Ilie Len Asp ACA ATA CAT TAC Thr Ilie Asp Tyr ACA CCC CTT GAG Arg Ciy Leu Ciu

AGC

Ar g 490 AAA GC GAG GAG Lys Vai Asp Gin GMA TGT MG A Gin Ser Asn Lys GTA MAC CCC CCC Vai Lys Gly Arg 1491 1539 1587 CCC CMA TCA ACT Arg Gin Set Thr AAA ACT AAA CTA Lys Thr Lys Leu CAT CCC ATT GAG Asp Aia Ile Gin ACC ACC Arg Thr 520 TCT CAT MAT Ser Asp Asn CCC CCA ACA AG Cly Ala Thr Arg ACT MG CGTC MAC Set Asri Val Lys AAA CCC TTC Lys Pro Len 535 1635 'WO 93/14211 W093/4211PCT/GB93/OO 19 ACA AAC A.AG Thr Asn Lys 540 AGA MAG GCT TCT Arg Lys Ala Ser ACG CAC AAG ATT Thr Asp Lys Ile CCC GTA AAT Ala Val Asn 1683 AGC AGA Ser Arg 555 GCT CGA TTG AAA Gly Arg Leu Lys TCC TTA ACA CAT Ser Leu Thr Asp ATA ACT GTG MAC Ile Thr Val Asn ACA MAC MAG GAT Thr Asn Lys Asp TTG ATT GTC GTG Leu Ile Val Val TGT TCT TCC MAG Cys Ser Set Lys 1731 1779 1827 TTT GTA TTG CTT Phe Val Leu Leu GTG ATG GMA GCC Val Met Glu Ala AGA CGA CTA ACT Arg Arg Leu Ser TTG CAT Leu Asp 600 TCC GMA ACT Set Glu Thr ATA MA CC Ile Lys Ala 620 CMA TCT TCC MAC Gin Ser Set Asn CAT CGA ATG ATA Asp Gly Met Ile TCT ATT ACC Set Ile Thr 615 ACT CTG AT'- Set Val Ile 1875 1923 MAG TCC MCG GGA Lys Cys Lys Gly MCG GTT CCA TCA Lys Val Ala Set AAA CMA Lys Gin 635 GCT CTT GAG A Ala Leu Gin Lys

CTT

Val 640 ACT ATC MAG TCT Thr Met Lys Ser TGMCGTTGAT TTATGCTCAC 1976 TATCTATAGC TACCTTTTCT CTAAMTT TGTATTCATA ACTTTTGCTA AGTMATTTGC ACCGCTTTTC CMCGTAGTTC AGATCMATM AAAAAAAMA 2036 2075 WO093/14211 IN4FORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 644 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: PCr/G B93/OOO 19 Met Ala Thr Gly Ile Gin Asn Gin Lys Ile Val Pro Giu Asn Leu Lys Gin Phe Trp Gly Phe Glu Leu Giu Leu Arg Pro Phe Phe Pro Gly 130 His Arg 145 Ala Ser Giu Leu His Ile Pro Asn 210 Giu Aia 225 Le u Ser Tyr As n Ty r Th r Le u 115 Arg Ala Ile Gly Ly s 195 Ty r Le u Ala As n As n Gin Giu Ala 100 Val Thr Asp Gin Ala 180 Th r Vali Giu Ile Ser Giy Asp Ser Ala Cys Le u Thr Th r 165 Th r Ser Ser His Ala Val Asp Gin 70 L~eu Leu Met Ala Ly s 150 Val Giu Phe Asn Ala 230 Val Ala Ile 55 Le u Ser Ser Ser Arg 135 Val Val Le u Le u Ser 215 Asn Arg Gin 40 Lys Gly Le u Pro Phe 120 As n Phe Cys Val Asp 200 Ile Ile Ser 25 Pror Th r Le u Gly Giu 105 11ie Gin Ser Phe Pro 185 Ser Th r Pro Ile Gly Arg Gin Giu 90 Asp Phe Ala Arg Pro 170 Giu Pro Asn Gin Val Ly s Arg 75 Th r Le u Asn Val S er 155 Ty r Asp Ala Asn Trp Le u Th r Ser Asn Th r Ile Trp 140 Le u Ser Le u Th r Asn 220 Ser Glu Vali Asp Th r Asp Gly 125 Le u Le u Giu Asn Val 205 Asp Tyr Trp Gin Gin Gin Ala 110 Gin Cys Ala Gly Leu 190 Pro Le u Ala Gly Ser Leu Ala Giu Gly Asn Lys Val 175 Ile Ly s Ile Arg Ile Asp Val Arg Ly s Trp Leu Ala Ser 160 Val Gin Ile Cys Giu Asn Asp Leu Asp Gin Leu WO93/14211 LeU Asn Cys Pro PC1'/GB93/OO1 9 Thr Asn Ilie Cys Ser Pro Asp Asn Ser Leu Asp 250 255 Asp Ile Al a Ser 305 Lys As n Gin Gly As n 385 Gin Asn Ala Arg Leu 465 Th r Ser Lys Arg Phe As n Ile 290 Gin Giy Gin Gly Pro 370 Ly s Ar g Ser Ly s Lys 450 Val Ile Asn Le u Thr 530 Asp 260 Giu As n His Pro Ile 340 Le u Phe Gly Leu Le u 420 Th r Arg Ser Tyr Met 500 Asp Asn Asp 245 Asn Va I Cys Giu Giu 325 Giu Ser Arg S er Ly s 405 Asp Ala Glu Gly Le u 485 Val1 Ala Val Le u Pro Leu As n 310 Th r As n As n As n Ser 390 Ly s Ala Asp Ly s Gly 470 Arg Lys 11ie Ly s Le u Gin As n 295 Le u Asn Th r Le u Gly 375 Gly Val Gly Clu Ile 455 Ly s Gly Gly Giu Ly s 535 Ile Asp 265 Thr Gin 280 Ser Ser Giu Ser Asn Cys Giy Val 345 Leu Lys 360 Asn Arg Thr His Leu Phe Lys Gin 425 Ile Asp 440 Asn Giu Val Asp Leu Glu Arg Gly 595 Arg Thr 520 Pro Leu Glu Ser Met Phe Met 330 Gin Ser Giu Val Giu 410 Lys Arg Arg Ly s Arg 490 Arg Ser Th r Ser Trp As n Ala 315 His Giy Ser Ser Pro 395 Val1 Giy As n Phe Val 475 Ly s Gl1u Asp Asn Asn Pro Ser 300 Pro Ser Asp His Ser 380 Arg Ala Asn His Met 460 Ser Val Ser Asn Lys 540 Le u Phe 285 Ser Leu Th r Glu Gin 365 Phe Ser Arg Ser Val 445 Ile Ile Asp Th r Tyr 525 Arg Ala 270 Met Asp Ser Gin Val 350 Leu ValI Gly Met Asp 430 Le u Le u Le u Giu Th r 510 Giy Ly s Giu Asp Cys Asp Lys 335 His Val1 Ser Th r His 415 Cys Ser Al a Asp Le u 495 Ly s Ala Ala Gly Asp Ile Gly 320 Cys Tyr Le Li Trp Ser 400 Glu Le u Glu Ser His 480 Giu Th r Th r Ser *WO 93/14211 PCT/GB93/00019 Asp Thr Asu Lys Ile Gly Ala Val Asn Ser Arg Cly Arg Leu Lys Asp 545 550 555 560 Ser Leu Thr Asp Asn Ile Thr Val Asn Ile Thr Asn Lys Asp Val Leu 565 570 575 Ile Val Val Thr Cy; Ser Ser Lys Glu Phe Val Leu Leu Glu Val Met 580 585 590 Glu Ala Val Arg Arg Leu Ser Leu Asp Ser Glu Thr Val Gin Ser Ser 595 600 605 Asn Arg Asp Gly Met Ile Ser Ile Thr Ile Lys Ala Lys Cys Lys Gly 610 615 620 Leu Lys Val Ala Ser Ala Ser Val Ile Lys Gin Ala Leu Gin Lys Val 625 630 635 640 Thr Met Lys Ser INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 54 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Mouse myogenin (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Asp Arg Arg Arg Ala Ala Thr Leu Arg Glu Lys Arg Arg Leu Lys Lys 1 5 10 Val Asn Clu Ala Phe Glu Ala Leu Lys Arg Ser Thr Leu Asn Pro Asn 25 Gin Arg Leu Pro Lys Val Glu Ile Leu Arg His Ala lIe Gin Tyr lle 40 Glu Arg Leu Gin Ala Leu WO 93/14211 PCT/GB93/00019 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 52 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Yeast Cbfl (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Gin Arg Lys Asp Ser His Lys Glu Val Glu Arg Arg Arg Arg Glu Asn 1 5 10 Ile Asn Thr Ala Ile Asn Val Leu Ser Asp Leu Ile Pro Val Arg Glu 25 Ser Ser Lys Ala Ala lie Leu Ala Arg Ala Ala Glu Tyr Ile Gin Lys 40 Leu Lys Glu Thr WO 93/14211 PCT/GB93/00019 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 55 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: AP-4 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Ile Arg Arg Glu Ile Ala Asn Ser Asn Glu Arg Arg Arg Met Gin Ser 1 5 10 Ile Asn Ala Gly Phe Gin Ser Leu Lys Thr Leu Ile Pro His Thr Asp 25 Gly Glu Lys Leu Ser Lys Ala Ala Ile Leu Gin Gin Thr Ala Glu Tyr 40 Ile Phe Ser Leu Glu Gin Glu WO 93/14211 PCT/GB93/00019 INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 56 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: N-myc (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Glu Arg Arg Arg Asn His Asn lie Leu Glu Arg Gin Arg Arg Asn Asp 1 5 10 Leu Arg Ser Ser Phe Leu Thr Leu Arg Asp His Val Pro Glu Leu Val 25 Lys Asn Glu Lys Ala Ala Lys Val Val Ile Leu Lys Lys Ala Thr Glu 40 Tyr Val His Ser Leu Gin Ala Glu *WO 93/14211 PCT/GB93/00019 INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 56 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: L-myc (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Thr Lys Arg Lys Asn His Asn Phe Leu Glu Arg Lys Arg Arg Asn Asp 1 5 10 Leu Arg Ser Arg Phe Leu Ala Leu Arg Asp Gln Val Pro Thr Leu Ala 25 Ser Cys Ser Lys Ala Pro Lys Val Val Ile Leu Ser Lys Ala Leu Glu 40 Tyr Leu Gin Ala Leu Val Gly Ala WO 93/14211 PCT/GB93/00019 INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 57 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Human E3 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Gin Lys Lys Asp Asn His Asn Leu Ile Glu Arg Arg Arg Arg Phe Asn 1 5 10 Ile Asn Asp Arg lie Lys Glu Leu Gly Thr Leu lle Pro Lys Ser Ser 25 Asp Pro Glu Met Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val 40 Asp Tyr Ile Arg Lys Leu Gin Lys Glu 'WO 93/142" 1 PCT/GB93/00019 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 53 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Maize R-S (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Ser Ala Thr Lys Asn His Val Met Ser Glu Arg Lys Arg Arg Glu Lys 1 5 10 Leu Asn Glu Met Phe Leu Val Leu Lys Ser Leu Leu Pro Ser Ile His 25 Arg Val Asn Lys Ala Ser lie Leu Ala Glu Thr Ile Ala Tyr Leu Lys 40 Glu Leu Gln Arg Arg WO 93/14211 PCT/GB93/00019 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 53 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Maize Lc (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Thr Gly Thr Lys Asn His Val Met Ser Glu Arg Lys Arg Arg Glu Lys 1 5 10 Leu Asn Glu Met Phe Leu Val Leu Lys Ser Leu Leu Pro Ser lie His 25 Arg Val Asn Lys Ala Ser Ile Leu Ala Glu Thr Ile Ala Tyr Leu Lys 40 Glu Leu Gln Arg Arg ,WO 93/14211 PCT/GB93/00019 INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 53 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Maize B (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Asn Gly Ala Lys Asn His Val Met Ser Glu Arg Lys Arg Arg Glu Lys 1 5 10 Leu Asn Glu Met Phe Leu Val Leu Lys Ser Leu Val Pro Ser lie His 25 Lys Val Asp Lys Ala Ser lle Leu Ala Glu Thr Ile Ala Tyr Leu Lys 40 Glu Leu Gin Arg Arg 'WO 93/14211 PCT/GB:, "'0019 INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 53 amino acids TYPE: amino acid TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (iii) HYPOTHETICAL: NO FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: ORGANISM: Antirrhinum majus (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Glu Ile Asp Arg Asn His Val Leu Ser Glu Arg Lys Arg Arg Glu Lys 1 5 10 Ile Asn Glu Arg Phe Met Ile Leu Ala Ser Leu Val Pro Ser Gly Gly 25 Lys Val Asp Lys Val Ser Ile Leu Asp His Thr Ile Asp Tyr Leu Arg 40 Gly Leu Glu Arg Lys

Claims (12)

1. A method of regulating the expression of one or more anthocyanin pigment genes in a plant which comprises the steps of transforming plant tissue with an expression vector comprising a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which shows substantial homology with the DEL protein as shown in SEQ ID NO 1 or 2 and which is capable of regulating expression of one or more plant genes involved in pigment biosynthesis, the said DNA segment being under the control of a promoter upstream of and operably linked thereto and regenerating from the transformed tissue plants showing altered anthocyanin pigmentation.

2. A method as claimed in claim 1 wherein the plant belongs to the dicotyledoneae.

3. A method as claimed in claim 1 or 2 wherein the DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 is a protein having an amino acid sequence which is at least preferably at least 90%, more preferably at least 98% homologous with the DEL protein as shown in SEQ ID NO 1 or 2.

4. A plant having a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which shows substantial homology with the DEL protein as shown in SEQ ID NO 1 or 2 incorporated into its genome or plant propagation material of such a plant.

5. A plant as claimed in claim 4 wherein the plant belongs to the dicotyledoneae. ;R A 42 SPCT internatlioal Appiction i 'TO .y PGTIG3/ 9 300019 14 FEBRUARY 1994

6. A plant as claimed in claim 4 or 5 wherein the DNA se gment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 is a protein having an amino acid sequence which is at least preferably at least 90%, more preferably at least 98% homologous with the DEL protein as shown in SEQ ID NO 1 or 2.

7. A DNA molecule encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO's 1 or 2 or a protein having an amino acid sequence which is at least preferably at least 90%, more preferably at least 98% homologous with the DEL protein as shown in SEQ ID NO 1 or 2 and which is capable of regulating expression of one or more plant genes involved in pigment biosynthesis.

8. Use of the DNA molecule as claimed in claim 7 or the protein encoded thereby to isolate a DNA molecule encoding a protein having the amino acid sequence which shows substantial homology with the DEL protein as shown in SEQ ID NO 1 or 2 from other plant species.

9. A construct which comprises a transposon having cloned therein a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 98% homologous with the DEL protein as shown in SEQ ID NO 1 or 2, the said DNA segment being under the control of a minimal promoter upstream of and operably linked thereto. A method of trapping a promoter/enhancer which comprises the steps of transforming a plant with the construct as claimed in claim 9 and deriving from the transformed plant further plants having a phenotype showing altered anthocyanin pigmentation.

11. A method for isolating a trapped promoter/enhancer from a plant which has been transformed with the construct as SOS- 43 LU" n u mm/D 33100019 14 FEBRUARY q,1 claimed in claim 9 which comprises reisolating the construct from said plant together with sequences adjacent thereto. 3" A method of expressing a gene of interest in a plant, which comprises transforming a cell of said plant with a first construct having said gene of interest under the control of a first promoter, which first promoter is that of an anthocyanin gene, upstream of and operably linked thereto, the said plant having incorporated into its genome a DNA segment encoding a protein having the amino acid sequence of the DEL protein as shown in SEQ ID NO 1 or 2 or a protein having an amino acid sequence which is at least 80%, preferably at least 90%, more preferably at least 98% homologous with the DEL protein as shown in SEQ ID NO 1 or 2 under the control of a second promoter upstream of and operably linked thereto, or the said plant being co-transformed with a second construct which comprises said DNA segment under the control of a third promoter, which third promoter may be the same or different to the second promoter, upstream of and operably linked thereto, or the said first construct optionally including the said DNA segment under the control of said second or third promoter upstream of and operably linked thereto if the said plant does not have incorporated into its genome the said DNA segment or is not co-transformed with the said second construct, and deriving from the transformed plant further plants expressing said gene of interest.

13. The method as claimed in claim 12 wherein the said second promoter for the said DNA segment is a promoter/enhancer isolated by the method as claimed in claim 11.

14. A method of expressing a gene of interest in a plant which comprises transforming said plant with the construct as claimed in claim 9, deriving from the transformed plant further plants having a phenotype showing altered anthocyanin pigmentation, reisolating from said plant the said construct y ."44 Q:\Ol'l\ JI 1i 64541.127 20/'96 together with sequences adjacent thereto, replacing said DNA segment in said construct with a gent of interest to form a new construct and transforming said plant with said new construct. A method of regulating the expression of one or more anthocyanin pigment genes in a plant according to any one of claims 1 to 3 or a plant having a DNA segment encoding a protein having the amino acid sequence of the DEL protein according to claim 4 or 5 or 6 or a DNA molecule encoding a protein having the amino acid sequence of the DEL protein according to claim 7 or the use of such a protein according to claim 8 or a method of trapping a promoter/enhancer according to claim 10 or 11 or a method of expressing a gene of interest in a plant according to claim 12 or 13 or 14 substantially as hereinbefore described with reference to the examples and/or figures. o i C S o C *o•