WO1993014211A1

WO1993014211A1 - Regulation of plant genes

Info

Publication number: WO1993014211A1
Application number: PCT/GB1993/000019
Authority: WO
Inventors: Enrico Coen; Rosemary Carpenter; Justin Goodrich; Roger FREEDMAN
Original assignee: John Innes Foundation
Priority date: 1992-01-09
Filing date: 1993-01-08
Publication date: 1993-07-22
Also published as: EP0620855A1; AU3645493A; AU671272B2; JPH07506000A

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 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.

Description

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, Madison, 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 CI 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 J . , 6 , 3553- 3558 (1987) and Ludwig et al, Proc . Natl . Acad . Sci . U . S .A . , 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 ma jus 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, i-bid 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 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. 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 80%, 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 90%, 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 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

80%, 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 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 shows substantial homology 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. 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 proteins 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 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 SEQ ID NO' s 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 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 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 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 shows substantial homology 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 (i.e. the promoter is at the 3' end of the 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. 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 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 probes.

FIGURE 7 - Plasmids pBJIMM15, pBJIMM21 and pBJIMM24. 1. Isolation of the 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 deI-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 dei-602 mutant and revertant plants was digested with restriction enzymes and probed with the various transposable elements isolated from A . majus . 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 dei-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 stock showed a wild-type band of 6.2 kb; and plants homozygous for a stable del allele, del-8, gave a 2.4 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/Hindlll fragment of the Tam2 clone pRH2, provided by Enno Krebbers and Hans Sommer. These plants were obtained in the F₂ 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 (E), Hindlll (H) and Bgrlll (B).

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 602, and the origin of the probe are shown below. Thick 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 μg 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 Tarn 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 (Lüttke et al., EMBO J ., 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 program (Lipman and Pearson, Science, 227, 1435-1441 (1985) revealed a strong homology between 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 carboxy terminus, residues 438-497 were 60% identical. The latter 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 μE3.

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 α 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 1). Northern analysis of RNA from del-602 flowers revealed various aberrant-sized transcripts (Figure 4a), which could result from the Tam2 insertion interfering with 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 mutation. Expression was also detected in the lobes, as was predicted from genetic interactions described previously (Almeida et al., ibid) . As expected, no wild- type 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 wild- type 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, 1 , 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. (a) 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 3μg of polyA⁺RNA. Note the hybridizing band at 2.5 kb. (b) 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 1), 17-20 nodes above (lane 2), 13-16 nodes above (lane 3), 9-12 nodes above (lane 4), 5-8 nodes above (lane 5), 1-4 nodes above (lane 6), 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) ) . (c) RNA from wild-type flower buds (1-5 nodes above first fully open flower) dissected into three parts: the base of corolla tubes (BT), the rest of the tube (RT), and the lobes (L) as indicated above the autoradiogram, probed with the del cDNA clone pJAM 121. (d) RNA from leaves (L), stems (S), bracts (B), sepals (Se), petals (P), stamens (St), styles (Sty) and carpels (Ca), probed with the del cDNA clone pJAM 121. Each lane of (b), (c) and (d) contains 10μg 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°C.

Nucleotide and predicted amino acid sequence of del cDNAs is shown in Figure 1 and SEQ ID NO 1. The predicted 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)) (ψ = L,I,V,M). 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. Biol., 1=1, 127-130 (1991)) and mouse myogenin (Edmondsen and Olson, Genes Dev . , 3, 628-640 (1989)).

cDNA was synthesised from 3μgpolyA⁺RNA extracted from wild-type flower buds (1-4 nodes above first fully opened flower), and cloned into the EcoRI site of ΛNM1149 using Amersham kits. A library of 10⁵ 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 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 confirm that the two sequences were contiguous, an intact clone, pJAM 121, was isolated by PCR amplification of cDNA ends (3' RACE) (Frohman et al., Proc. Natl. Acad. Sci. U.S.A., 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 ³⁵S labelled RNA probes. (a) Tube tissue under light (left) and dark field (right). The inner epidermis (I), mesophyll (M) and outer epidermis (O) 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 epidermis, 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 eds ) Oxford University Press (1991). A lkb 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. ³⁵S RNA probes were synthesised using lμg of linearised plasmid as template, t7 polymerase and 50 μCi of ³⁵S 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°C. No signal above background was observed using control sense- strand 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 0). The seeds were allowed to germinate and grow for 10 days (day 10).

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, pBJIMM15, 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 P.N.A.S., 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' terminator sequence, pBJIMM21 carries a copy of the del gene derived from a genomic fragment again driven by the 35S 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 J., 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 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 showing 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 pBJIMMlδ and pBJIMM21 cultures. On day 0 10 ml volumes of Luria broth were inoculated with Agrobacterium 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 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 pBJIMM15 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 sub- epidermal 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. SEQUENCE LI STI NG GENERAL INFORMATION :

( i ) 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

(ii) TITLE OF INVENTION: Regulation of Plant Genes

(iii) NUMBER OF SEQUENCES: 12

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(v) CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/GB93/

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/818,570

(B) FILING DATE: 09-JAN-1992

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2075 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Antirrhinum Majus

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 25..1956

(ix) FEATURE:

(A) NAME/KEY: insertion_seq

(B) LOCATION: (573^574)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GTAGAGAGGA GAGAGGATTC AAGA ATG GCT ACT GGT ATC CAA AAC CAA AAG 51

Met Ala Thr Gly Ile Gln Asn Gln Lys

1 5

ATA GTG CCT GAG AAT TTG AGG AAG CAA CTT GCT ATT GCT GTG AGA AGT 99 Ile Val Pro Glu Asn Leu Arg Lys Gln Leu Ala Ile Ala Val Arg Ser

10 15 20 25

ATC CAA TGG AGT TAT GCA ATT TTC TGG TCC AAT TCA GTT GCA CAA CCA 147 Ile Gln Trp Ser Tyr ALa Ile Phe Trp Ser Asn Ser Val Ala Gln Pro

30 35 40

GGG GTC TTG GAG TGG GGT GAT GGG TTC TAC AAT GGA GAT ATT AAA ACT 195 Gly Val Leu Glu Trp Gly Asp Gly Phe Tyr Asn Gly Asp Ile Lys Thr

45 50 55

CGA AAA ACT GTA CAA TCT GTC GAA TTG AAT CAA GAT CAG CTG GGA TTG 243 Arg Lys Thr Val Gln Ser Val Glu Leu Asn Gln Asp Gln Leu Gly Leu

60 65 70

CAG AGA AGT GAT CAA TTG AGA GAA CTT TAT GAG TCT CTT TCA CTT GGT 291 Gln Arg Ser Asp Gln Leu Arg Glu Leu Tyr Glu Ser Leu Ser Leu Gly

75 80 85 GAA ACC AAC ACA CAA GCT AAA AGG CCT ACT GCT GCA TTA TCA CCA GAA 339

Glu Thr Asn Thr Gln Ala Lys Arg Pro Thr Al a Ala Leu S er Pro G l u

90 95 100 105

GAC CTC ACT GAT GCT GAG TGG TTT TTC TTG GTT TGC ATG TCT TTC ATA 387

Asp Leu Thr Asp Ala Glu Trp Phe Phe Leu Val Cys Met Ser Phe Ile

110 115 120

TTC AAT ATT GGC CAA GGG TTG CCT GGA AGA ACA TTA GCA CGA AAT CAA 435

Phe Asn Ile Gly Gln Gly Leu Pro Gly Arg Thr Leu Ala Arg Asn Gln

125 130 135

GCA GTA TGG CTA TGC AAC GCT CAT CGT GCG GAC ACC AAA GTT TTC TCG 483

Ala Val Trp Leu Cys Asn Ala His Arg Ala Asp Thr Lys Val Phe Ser

140 145 150

CGT TCT TTG CTT GCA AAG AGT GCG TCA ATT CAG ACA GTT GTG TGC TTT 531

Arg Ser Leu Leu Ala Lys Ser Ala Ser Ile Gln Thr Val Val Cys Phe

155 160 165

CCA TAT TCA GAA GGT GTA GTT GAG CTG GGA GCA ACA GAG CTA GTA CCG 579

Pro Tyr Ser Glu Gly Val Val Glu Leu Gly Ala Thr Glu Leu Val Pro

170 175 180 185

GAG GAT TTG AAT CTA ATC CAG CAT ATA AAA ACT TCA TTC TTG GAC AGT 627

Glu Asp Leu Asn Leu Ile Gln His Ile Lys Thr Ser Phe Leu Asp Ser

190 195 200

CCT GCC ACC GTT CCC AAG ATT CCC AAC TAT GTC TCC AAC AGT ATT ACA 675

Pro Ala Thr Val Pro Lys Ile Pro Asn Tyr Val Ser Asn Ser Ile Thr

205 210 215

AAC AAC AAT GAC CTC ATT TGT GAA GCG CTT GAA CAT GCT AAT ATA CCA 723

Asn Asn Asn Asp Leu Ile Cys Glu Ala Leu Glu His Ala Asn Ile Pro

220 225 230

GAA AAC GAT CTT GAT CAG CTT TTG AAT TGT CCA GAC ACG AAC ATA TGT 771

Glu Asn Asp Leu Asp Gln Leu Leu Asn Cys Pro Asp Thr Asn Ile Cys

235 240 245

TCT CCT GAT AAC AGT TTG GAT GAC TTT GCA GAC AAT TTA CTC ATA GAC 819

Ser Pro Asp Asn Ser Leu Asp Asp Phe Ala Asp Asn Leu Leu Ile Asp

250 255 260 265

GAA TCG AAT TTG GCA GAA GGC ATC AAT GGG GAG GTT CCT CAA ACA CAA 867

Glu Ser Asn Leu Ala Glu Gly Ile Asn Gly Glu Val Pro Gln Thr Gln

270 275 280

AGC TGG CCT TTC ATG GAT GAT GCA ATC AGC AAT TGT CTC AAT AGT TCT 915

Ser Trp Pro Phe Met Asp Asp Ala Ile Ser Asn Cys Leu Asn Ser Ser

285 290 295

ATG AAT TCT AGT GAC TGT ATA TCT CAA ACT CAT GAA AAT CTA GAG TCT 963

Met Asn Ser Ser Asp Cys Ile Ser Gln Thr His Glu Asn Leu Glu Ser

300 305 310 TTT GCT CCA CTT TCT GAT CGA AAA GGC CCA CCG GAG ACG AAT AAT TGT 1011 Phe Ala Pro Leu Ser Asp Gly Lys Gly Pro Pro Glu Thr Asn Asn Cys

315 320 325

ATG CAC AGC ACT CAA AAA TGC AAT CAG CAG ATA GAA AAC ACG GGT GTC 1059 Met His Ser Thr Gln Lys Cys Asn Gln Gln Ile Glu Asn Thr Gly Val

330 335 340 345

CAA GGC GAT GAG GTC CAT TAT CAA GGG GTA CTT TCC AAT CTT TTG AAG 1107 Gln Gly Asp Glu Val His Tyr Gln Gly Val Leu Ser Asn Leu Leu Lys

350 355 360

AGT TCC CAT CAG TTG GTT CTT GGT CCC TAC TTC AGA AAT GGG AAT AGA 1155 Ser Ser His Gln Leu Val Leu Gly Pro Tyr Phe Arg Asn Gly Asn Arg

365 370 375

GAA TCA AGC TTC GTT AGT TGG AAC AAG GAT GGA TCG TCG GGT ACT CAT 1203 Glu Ser Ser Phe Val Ser Trp Asn Lys Asp Gly Ser Ser Gly Thr His

380 385 390

GTT CCC CGA AGC GGA ACC TCA CAA AGA TTT CTG AAG AAA GTA CTT TTT 1251 Val Pro Arg Ser Gly Thr Ser Gln Arg Phe Leu Lys Lys Val Leu Phe

395 400 405

GAA GTA GCT AGA ATG CAT GAA AAC TCC AGG CTT GAT GCT GGT AAA CAA 1299 Glu Val Ala Arg Met His Glu Asn Ser Arg Leu Asp Ala Gly Lys Gln

410 415 420 425

AAG GGC AAC AGT GAC TGC CTT GCA AAG CCA ACG GCT GAT GAA ATT GAT 1347 Lys Gly Asn Ser Asp Cys Leu Ala Lys Pro Thr Ala Asp Glu Ile Asp

430 435 440

AGA AAC CAC GTC TTG TCA GAG AGA AAA CGC AGA GAG AAA ATA AAC GAA 1395 Arg Asn His Val Leu Ser Glu Arg Lys Arg Arg Glu Lys Ile Asn Glu

445 450 455

CGG TTT ATG ATT CTT GCA TCC CTA GTC CCA TCC GGT GGC AAG GTT GAC 1443 Arg Phe Met Ile Leu Ala Ser Leu Val Pro Ser Gly Gly Lys Val Asp

460 465 470

AAA GTA TCA ATA CTA GAC CAT ACA ATA GAT TAC TTG AGA GGG CTT GAG 1491 Lys Val Ser Ile Leu Asp His Thr Ile Asp Tyr Leu Arg Gly Leu Glu

475 480 485

AGG AAA GTC GAC GAG CTG GAA TCT AAC AAA ATG GTA AAG GGC CGG GGG 1539 Arg Lys Val Asp Glu Leu Glu Ser Asn Lys Met Val Lys Gly Arg Gly

490 495 500 505

CGG GAA TCA ACT ACA AAA ACT AAA CTA CAC GAT GCC ATT GAG AGG ACC 1587 Arg Glu Ser Thr Thr Lys Thr Lys Leu His Asp Ala Ile Glu Arg Thr

510 515 520

TCT GAT AAT TAT GGC GCA ACA AGG ACA AGT AAC GTC AAG AAA CCG TTG 1635 Ser Asp Asn Tyr Gly Ala Thr Arg Thr Ser Asn Val Lys Lys Pro Leu

525 530 535 ACA AAC AAG AGA AAG GCT TCT GAT ACG GAC AAG ATT GGA GCC GTA AAT 1683 Thr Asn Lys Arg Lys Ala Ser Asp Thr Asp Lys Ile Gly Ala Val Asn

540 545 550

AGC AGA GGT CGA TTG AAA GAT TCC TTA ACA GAT AAT ATA ACT GTG AAC 1731 Ser Arg Gly Arg Leu Lys Asp Ser Leu Thr Asp Asn Ile Thr Val Asn

555 560 565

ATT ACA AAC AAG GAT GTG TTG ATT GTC GTG ACT TGT TCT TCC AAG GAG 1779 Ile Thr Asn Lys Asp Val Leu Ile Val Val Thr Cys Ser Ser Lys Glu

570 575 580 585

TTT GTA TTG CTT GAA GTG ATG GAA GCC GTA AGA CGA CTA AGT TTG GAT 1827 Phe Val Leu Leu Glu Val Met Glu Ala Val Arg Arg Leu Ser Leu Asp

590 595 600

TCC GAA ACT GTT CAA TCT TCC AAC AGA GAT GGA ATG ATA TCT ATT ACC 1875 Ser Glu Thr Val Gln Ser Ser Asn Arg Asp Gly Met Ile Ser Ile Thr

605 610 615

ATA AAA GCC AAG TGC AAG GGA TTG AAG GTT GCA TCA GCA AGT GTG ATC 1923 Ile Lys Ala Lys Cys Lys Gly Leu Lys Val Ala Ser Ala Ser Val Ile

620 625 630

AAA CAA GCT CTT CAG AAA GTT ACT ATG AAG TCT TGAAGTTGAT TTATGCTCAC 1976 Lys Gln Ala Leu Gln Lys Val Thr Met Lys Ser

635 640

TATCTATAGC TAGCTTTTGT GTAAAAAATT TGTATTCATA ACTTTTGCTA AGTAATTTGC 2036

AGGGCTTTTC CAAGTAGTTC AGATCAATAA AAAAAAAAA 2075

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 644 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ala Thr Gly Ile Gln Asn Gln Lys Ile Val Pro Glu Asn Leu Arg 1 5 10 15

Lys Gln Leu Ala Ile Ala Val Arg Ser Ile Gln Trp Ser Tyr Ala Ile

20 25 30

Phe Trp Ser Asn Ser Val Ala Gln Pro Gly Val Leu Glu Trp Gly Asp

35 40 45

Gly Phe Tyr Asn Gly Asp Ile Lys Thr Arg Lys Thr Val Gln Ser Val 50 55 60

Glu Leu Asn Gln Asp Gln Leu Gly Leu Gln Arg Ser Asp Gln Leu Arg 65 70 75 80

Glu Leu Tyr Glu Ser Leu Ser Leu Gly Glu Thr Asn Thr GLn Ala Lys

85 90 95

Arg Pro Thr Ala Ala Leu Ser Pro Glu Asp Leu Thr Asp Ala Glu Trp

100 105 110

Phe Phe Leu Val Cys Met Ser Phe Ile Phe Asn Ile Gly Gln Gly Leu

115 120 125

Pro Gly Arg Thr Leu Ala Arg Asn Gln Ala Val Trp Leu Cys Asn Ala 130 135 140

His Arg Ala Asp Thr Lys Val Phe Ser Arg Ser Leu Leu Ala Lys Ser 145 150 155 160

Ala Ser Ile Gln Thr Val Val Cys Phe Pro Tyr Ser Glu Gly Val Val

165 170 175

Glu Leu Gly Ala Thr Glu Leu Val Pro Glu Asp Leu Asn Leu Ile Gln

180 185 190

His Ile Lys Thr Ser Phe Leu Asp Ser Pro Ala Thr Val Pro Lys Ile

195 200 205

Pro Asn Tyr Val Ser Asn Ser Ile Thr Asn Asn Asn Asp Leu Ile Cys 210 215 220

Glu Ala Leu Glu His Ala Asn Ile Pro Glu Asn Asp Leu Asp Gln Leu 225 230 235 240 Leu Asn Cys Pro Asp Thr Asn Ile Cys Ser Pro Asp Asn Ser Leu Asp 245 250 255

Asp Phe Ala Asp Asn Leu Leu Ile Asp Glu Ser Asn Leu Ala Glu Gly

260 265 270

Ile Asn Gly Glu Val Pro Gln Thr Gln Ser Trp Pro Phe Met Asp Asp

275 280 285

Ala Ile Ser Asn Cys Leu Asn Ser Ser Met Asn Ser Ser Asp Cys Ile 290 295 300

Ser Gln Thr His Glu Asn Leu Glu Ser Phe Ala Pro Leu Ser Asp Gly 305 310 315 320

Lys Gly Pro Pro Glu Thr Asn Asn Cys Met His Ser Thr Gln Lys Cys

325 330 335

Asn Gln Gln Ile Glu Asn Thr Gly Val Gln Gly Asp Glu Val His Tyr

340 345 350

Gln Gly Val Leu Ser Asn Leu Leu Lys Ser Ser His Gln Leu Val Leu

355 360 365

Gly Pro Tyr Phe Arg Asn Gly Asn Arg Glu Ser Ser Phe Val Ser Trp 370 375 380

Asn Lys Asp Gly Ser Ser Gly Thr His Val Pro Arg Ser Gly Thr Ser 385 390 395 400 Gln Arg Phe Leu Lys Lys Val Leu Phe Glu Val Ala Arg Met His Glu

405 410 415

Asn Ser Arg Leu Asp Ala Gly Lys Gln Lys Gly Asn Ser Asp Cys Leu

420 425 430

Ala Lys Pro Thr Ala Asp Glu Ile Asp Arg Asn His Val Leu Ser Glu

435 440 445

Arg Lys Arg Arg Glu Lys Ile Asn Glu Arg Phe Met Ile Leu Ala Ser 450 455 460

Leu Val Pro Ser Gly Gly Lys Val Asp Lys Val Ser Ile Leu Asp His 465 470 475 480

Thr Ile Asp Tyr Leu Arg Gly Leu Glu Arg Lys Val Asp Glu Leu Glu

485 490 495

Ser Asn Lys Met Val Lys Gly Arg Gly Arg Glu Ser Thr Thr Lys Thr

500 505 510

Lys Leu His Asp Ala Ile Glu Arg Thr Ser Asp Asn Tyr Gly Ala Thr

515 520 525

Arg Thr Ser Asn Val Lys Lys Pro Leu Thr Asn Lys Arg Lys Ala Ser 530 535 540 Asp Thr Asp Lys Ile Gly Ala Val Asn Ser Arg Gly 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 Cys 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 Gln 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 Gln Ala Leu Gln Lys Val 625 630 635 640

Thr Met Lys Ser

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) 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 15

Val Asn Glu Ala Phe Glu Ala Leu Lys Arg Ser Thr Leu Asn Pro Asn

20 25 30

Gln Arg Leu Pro Lys Val Glu Ile Leu Arg His Ala Ile Gln Tyr Ile

35 40 45

Glu Arg Leu Gln Ala Leu

50 (2) INFORMATION FOR SEQ ID NO: 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Yeast Cbf1

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Gln Arg Lys Asp Ser His Lys Glu Val Glu Arg Arg Arg Arg Glu Asn 1 5 10 15 Ile Asn Thr Ala Ile Asn Val Leu Ser Asp Leu Ile Pro Val Arg Glu

20 25 30

Ser Ser Lys Ala Ala Ile Leu Ala Arg Ala Ala Glu Tyr Ile Gln Lys

35 40 45

Leu Lys Glu Thr

50

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 55 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: AP-4

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ile Arg Arg Glu Ile Ala Asn Ser Asn Glu Arg Arg Arg Met Gln Ser 1 5 10 15 Ile Asn Ala Gly Phe Gln Ser Leu Lys Thr Leu Ile Pro His Thr Asp

20 25 30

Gly Glu Lys Leu Ser Lys Ala Ala Ile Leu Gln Gln Thr Ala Glu Tyr

35 40 45 Ile Phe Ser Leu Glu Gln Glu

50 55

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: N-myc

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Glu Arg Arg Arg Asn His Asn Ile Leu Glu Arg Gln Arg Arg Asn Asp 1 5 10 15

Leu Arg Ser Ser Phe Leu Thr Leu Arg Asp His Val Pro Glu Leu Val

20 25 30

Lys Asn Glu Lys Ala Ala Lys Val Val Ile Leu Lys Lys Ala Thr Glu

35 40 45

Tyr Val His Ser Leu Gln Ala Glu

50 55

INFORMATION FOR SEQ ID NO : 7 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 56 amino ac i ds

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) 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 15

Leu Arg Ser Arg Phe Leu Ala Leu Arg Asp Gln Val Pro Thr Leu Ala

20 25 30

Ser Cys Ser Lys Ala Pro Lys Val Val Ile Leu Ser Lys Ala Leu Glu

35 40 45

Tyr Leu Gln Ala Leu Val Gly Ala

50 55

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 57 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Human E3

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Gln Lys Lys Asp Asn His Asn Leu Ile Glu Arg Arg Arg Arg Phe Asn 1 5 10 15 Ile Asn Asp Arg Ile Lys Glu Leu Gly Thr Leu Ile Pro Lys Ser Ser

20 25 30

Asp Pro Glu Met Arg Trp Asn Lys Gly Thr Ile Leu Lys Ala Ser Val

35 40 45

Asp Tyr Ile Arg Lys Leu Gln Lys Glu

50 55

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 53 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) 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 15

Leu Asn Glu Met Phe Leu Val Leu Lys Ser Leu Leu Pro Ser Ile His

20 25 30

Arg Val Asn Lys Ala Ser Ile Leu Ala Glu Thr Ile Ala Tyr Leu Lys

35 40 45

Glu Leu Gln Arg Arg

50

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 53 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Maize Lc

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

Thr Gly Thr Lys Asn His Val Met Ser Glu Arg Lys Arg Arg Glu Lys 1 5 10 15

Leu Asn Glu Met Phe Leu Val Leu Lys Ser Leu Leu Pro Ser Ile His

20 25 30

Arg Val Asn Lys Ala Ser Ile Leu Ala Glu Thr Ile Ala Tyr Leu Lys

35 40 45

Glu Leu Gln Arg Arg

50

INFORMATION FOR SEQ ID NO : 11 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 53 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) 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 15

Leu Asn Glu Met Phe Leu Val Leu Lys Ser Leu Val Pro Ser Ile His

20 25 30

Lys Val Asp Lys Ala Ser Ile Leu Ala Glu Thr Ile Ala Tyr Leu Lys

35 40 45

Glu Leu Gln Arg Arg

50

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 53 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: NO

(v) FRAGMENT TYPE: internal

(vi) ORIGINAL SOURCE:

(A) 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 15 Ile Asn Glu Arg Phe Met Ile Leu Ala Ser Leu Val Pro Ser Gly Gly

20 25 30

Lys Val Asp Lys Val Ser Ile Leu Asp His Thr Ile Asp Tyr Leu Arg

35 40 45

Gly Leu Glu Arg Lys

50

Claims

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 80%, preferably at least 90%, more preferably at least 98% similar 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.

6. A plant as claimed in claim 4 or 5 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 80%, preferably at least 90%, more preferably at least 98% similar 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 80%, 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.

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 shows substantial homology 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.

10. 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 claimed in claim 9 which comprises reisolating the construct from said plant together with sequences adjacent thereto.

12. 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 shows substantial homology 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 8, 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.