METHODS FOR INCREASING THE ANTHOCYANIN CONTENT OF CITRUS FRUIT

Abstract

Methods are provided for making a citrus plant that is capable of producing fruit with increased levels of anthocyanins in the fruit when compared to a citrus fruit from a wild-type or control plant. Such methods involve increasing the expression of Ruby, an R2R3 Myb transcription factor, in a citrus plant, particularly in the fruit of a citrus plant. Further provided are citrus plants, citrus fruit, and citrus plant cells made by such methods, nucleic acid molecules and expression cassettes comprising nucleotide sequences that encode Ruby, and the Ruby proteins encoded thereby.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 13/749,774, filed Jan. 25, 2013 and claims the benefit of U.S. Provisional Patent Application No. 61/591,115, filed Jan. 26, 2012; both of which are herein incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS WEB

The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 429212SeqLst.txt, created on Jan. 24, 2013, and having a size of 68.4 kilobytes, and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of plant molecular biology, particularly the improvement of plants through the use of genetic engineering methods.

BACKGROUND OF THE INVENTION

In addition to their striking color, blood oranges are believed to have significant health-promoting properties, combining the high content of vitamin C, carotenoids and fiber of common blond oranges with the health-promoting properties of anthocyanin pigments (de Pascual-Theresa et al., 2010; Paradez-Lopez et al., 2010; Davies, 2007; Prior and Wu, 2006). The high anthocyanin content of blood oranges underpins their high antioxidant activity (Proteggente et al., 2011; Kelebek et al., 2008; Jayaprakasha and Patl, 2007; Rapisada et al., 1999). Consumption of blood orange juice has been shown to reduce oxidative stress in diabetic patients (Bottina et al., 2002), protect DNA against oxidative damage (Guarnieri et al., 2007; Riso et al., 2005) and may reduce cardiovascular risk factors more generally, as demonstrated for other high-anthocyanin foods (de Pascual-Theresa et al., 2010; Paradez-Lopez et al., 2010; Toufektsian et al., 2008). Recently, blood orange juice has been shown to limit the development of adipocytes and weight gain in mice and to confer resistance to obesity compared to blond orange juice or water (Titta et al., 2010). In a mouse model of obesity, blood orange juice consumption rescued almost completely the transcriptional reprogramming induced by a high fat diet.

Despite increasing consumer interest in their high nutritional quality, blood oranges do not have a global market, largely due to a lack of dependability of color development. All blood orange varieties require strong day-night thermal clines for intense color formation in fruit flesh, and varieties such as Moro, with the potential for high pigmentation, are strongly dependent on the prevailing climatic conditions during fruit ripening for full colour development. Post-harvest storage of fruit in the cold enhances pigmentation, but this is an expensive measure to ensure high levels of pigmentation, and can increase post-harvest losses (Crifò et al., 2011; Latado et al., 2008; Rapisada et al., 1999). The dependency of anthocyanin accumulation on environment means that the most reliable blood orange production, on a commercial scale, is limited to Italy, specifically to the Sicilian area around Mount Etna (Zarba and Pulvirenti, 2006) where it remains highly seasonal. Although blood oranges are grown in other countries, in some years entire harvests are lost due to non-optimal conditions during ripening of fruit, and when they are cultivated in Brazil or Florida (the largest producers of oranges worldwide), coloration is generally weak or absent and unreliable (Latado et al., 2008; Hodgson, 1967). To ensure a stable supply of blood oranges, improved oranges trees which can produce blood oranges with reliably high levels of anthocyanins under a variety of environmental conditions, are desired.

Anthocyanins are natural pigments found typically in red, purple and blue fruit and flowers (Winkel-Shirley, 2001). Many varieties of blood orange have been derived from old Italian varieties such as Doppio Sanguigno and include more recently-derived varieties such as Tarocco and Moro, which generally have higher levels of anthocyanin pigmentation of their fruit (FIG. 1). The history of these varieties is debated although authoritative texts suggest three independent derivations: one Italian/Sicilian from Doppio Sanguigno/Maltaise Sanguine, a second in Spain from Doblefina and a third from Shamouti Orange referred to as Shamouti Blood or Palestinian Blood Jaffa Orange (Hodgson, 1967). In the mid-19th century it was believed that blood oranges arose by bud mutation in the Mediterranean region following the introduction of sweet orange in the 16^th century (Holmes, 1924). More recently it was suggested that blood oranges originated much earlier in Asia (Hodgson, 1967; Chapot, 1963). The blood orange was first documented in Italy in ‘Hesperides’ by Ferrari (1646), a Jesuit scholar who wrote of an orange with purple-colored flesh that tasted strangely like a grape (Ferrari, 1646). Ferrari suggested that the blood orange was brought to Sicily by a Genoese missionary after a long journey which started in China. However, claims of Chinese poems referring to scarlet/red oranges, dating from the Tang period to more recent times, are probably based on mistranslation of the term ‘orange’ and likely refer to mandarins which never accumulate anthocyanins. Some definitively red oranges are portrayed in a picture by Bartolomeo Bimbi, a Florentine artist who painted the Medici Citrus collections early in the 18^th century.

BRIEF SUMMARY OF THE INVENTION

Methods are provided for making a citrus plant that is capable of producing fruit with increased levels of anthocyanins in the fruit when compared to a citrus fruit from a wild-type or other control plant. The methods of the present invention involve increasing the expression of Ruby in a citrus plant, particularly in the fruit of a citrus plant. Ruby is a novel R2R3 Myb transcription factor that regulates the expression of genes required for anthocyanin biosynthesis in citrus fruit as disclosed hereinbelow. In one embodiment, the methods comprise introducing into at least one citrus plant cell a polynucleotide construct comprising a promoter operably linked to nucleotide sequence encoding Ruby. Preferably, the promoter is capable of driving the expression of the nucleotide sequence encoding Ruby in a citrus fruit or part thereof, particularly the carpels or endocarp. The methods of the invention can further comprise regenerating a citrus plant comprising the polynucleotide construct.

In another embodiment, the methods comprise introducing into at least one citrus plant cell a polynucleotide construct comprising a promoter that is capable of driving the expression of an operably linked nucleotide sequence. In this embodiment, the citrus plant cell comprises stably incorporated in its genome a native or non-native nucleotide sequence encoding a functional Ruby protein. Such a method further comprises the use of homologous recombination methods that are known in the art to incorporate the introduced polynucleotide construct comprising a promoter in operable linkage with the nucleotide sequence encoding a functional Ruby protein, whereby the promoter is capable of driving the expression of the nucleotide sequence encoding a functional Ruby protein. Preferably, the promoter is capable of driving the expression of the nucleotide sequence encoding Ruby in a citrus fruit or part thereof, particularly the carpels or endocarp. The methods of the invention can further comprise regenerating a citrus plant comprising the polynucleotide construct.

Isolated nucleic acid molecules comprising the Ruby nucleotide sequences disclosed herein and fragments and variants thereof that encode functional Ruby proteins are further provided as wells as the Ruby proteins encoded thereby. Also provided are nucleic acid molecules comprising the retrotransposons, Tcs1 and Tcs2. Expression cassettes comprising a promoter operably linked to a nucleotide sequence encoding a Ruby protein are additional provided.

Additionally provided are citrus plants and citrus plant cell made by the methods disclosed herein as well as citrus fruit produced from such plants and food products derived from the citrus fruit including, for example, citrus fruit juice, beverages comprising citrus fruit juice (e.g., sodas, smoothies and other blended beverages). marmalades, and food colorants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Phenotypes and genotypes of orange varieties and hybrids: (A) Navelina R, r-2; (B) Doppio Sanguigno R^D-1, r-2; (C) Tarocco (CRA) R^D-1, r-2; (D) Moro (CRA) R^D-2, r-2; (E) OTA3 R^D-2, r-2; (F) OTA7 r-2, r-2; (G) OTA9 R^D-2, r-1; (H) OTA17 r-1, r-2; and (I) Jingxian R^D-3, r-2.

FIG. 2. Features of the protein encoded by the Ruby transcript. (A) Protein alignment of Ruby (CsRuby; SEQ ID NO: 2) with characterised members of the anthocyanin-specific family of Myb factors (available on the World Wide Web at multalin.toulouse.inra.fr/multalin/). Identical amino acids are shown in pink. The GenBank accession numbers of these proteins are as follows: PhAN2 (Petunia×hybrida), GenBank: AAF66727.1 (SEQ ID NO: 43); S1AN1 (Solanum lycopersicum), AAQ55181.1 (SEQ ID NO: 44); VvMYBA1 (Vitis vinifera), ABB87013.1 (SEQ ID NO: 45); IbMYB1 (Ipomoea batatas), BAF45118.1 (SEQ ID NO: 46); AtPAP1 (Arabidopsis thaliana), ABB03877.1 (SEQ ID NO: 47); AmROSEA1 (Antirrhinum majus), ABB83826.1 (SEQ ID NO: 48). Arrows indicate the region to which degenerated primers were designed. The signature motif for interaction with bHLH proteins within the R3 Myb DNA binding domain and the conserved motif defining members of R2R3 Myb subgroup 6 are boxed in grey. (B) Phylogenetic analysis showing that the Ruby transcription factor clusters with anthocyanin-specific members of subgroup 6 from the R2R3MYB family. Bootstrap values are shown if significant at or above the 90 percent confidence limit. Locus identifiers for Arabidopsis proteins: AtMYB3, At1g22640; AtMYB4, At4g38620; AtMYB6, At4g09460; AtMYB7, At2g16720; AtMYB8, At1g35515; AtMYB13, At1g06180; AtMYB14, At2g31180; AtMYB15, At3g23250; AtMYB32, At4g34990; AtMYB11, At3g62610; AtMYB12, At2g47460; AtMYB111, At5g49330; AtMYB113, At1g66370; AtMYB114, At1g66380; AtMYB75, At1g56650; AtMYB90, At1g66390; AtMYB123, At5g35550. Identifiers for other proteins: CsRuby, (this study, BankIt accession number: 1469607); NtMYB2, AB028649 (Nt, tobacco); DcMYB1, AB218778 (carrot); NtMYBJS1, AB236951; OsMYB4, Y11414 (rice); NaMYB8, GU451752 (Nicotiana attenuate); EgMYB 1, AJ576024 (eucalyptus); AmMYB308, P81393 (Am, Antirrhinum); AmMYB330, P81395; ZmMYB42, AM156908 (Zm, maize); PtMYB14, DQ399056 (Pinus taeda); ZmMYBP, U57002; S1MYB12, EU419748 (tomato); SbMYBY1, AY860968 (Sorghum bicolour); VvMYBF1, FJ948477 (Vv, grape); AmROSEA1, DQ275529; AmROSEA2, DQ275530; AmVENOSA, DQ275531; VvMYBA1, AB097923; VvMYBA2, AB097924; LeANT1, AY348870 (Lycopersicon esculentum); PhAN2, AF146702 (Petunia hybrida); ZmMYBC1, X06333; ZmMYBPL, L19494.

FIG. 3. Ruby is a regulator of anthocyanin biosynthesis. Constitutive expression of Ruby cDNA under the control of the 35S promoter in tobacco results in strong anthocyanin-based pigmentation in (A) undifferentiated callus and (B) regenerating shoots. (C) Coexpression of the bHLH regulatory genes Delila and Mutabilis increases further anthocyanin accumulation in mature leaves: (1) 35S:Ruby (2) 35S:Ruby×35S:Delila (3) 35S:Ruby×35S:Mutabilis (4) 35S:Rosea1×35S:Delila (5) 35S:Rosea1×35S:Mutabilis (D) Ruby partners more successfully with the bHLH protein Mutabilis than with the bHLH protein Delila to activate anthocyanin biosynthesis in tobacco. Anthocyanin accumulation in two seedlings of each genotype are shown. (E) Anthocyanin content in tobacco seedlings transformed with different regulatory genes.

FIG. 4. Expression analysis of Ruby. (A) Expression of Ruby in different tissues of blond Cadenera and blood Moro oranges. Albedo is the spongey white layer of the orange peel. The flavedo is the peripheral surface of the pericarp. (B) Expression of Ruby in diploid hybrids obtained crossing mandarin (C. clementina cv. Oroval) with C. sinensis cv. Moro (OMO series) or C. sinensis cv. Tarocco (OTA series). Hybrids lacking anthocyanins (OMO41,3,5, OTA 1,17,31,35) appear slightly different colours due to varying carotenoid levels in the fruit flesh.

FIG. 5 Genomic characterization of the Ruby locus. (A) Southern blot of genomic DNA from different blood and blond orange varieties and from hybrids between Tarocco and mandarin (OTA) and Moro and mandarin (OMO) digested with AseI and probed with a ³²P-labelled probe of the Ruby gene from 465 bp upstream of the ATG to the stop codon of the gene. Varieties: N=Navelina; T (UK)=Tarocco from Reeds (UK); J=Jingxian; DS=Doppio Sanguigno; C=Cadenera; O=Oroval Clementine (mandarin); T (I)=Tarocco from CRA, Italy; M (I)=Moro from CRA; OTA hybrids 3, 9, 14, and 20 are pigmented with anthocyanin; OMO hybrids 28 and 31 are pigmented with anthocyanin. Band 1 is the AseI fragment at the 5′ end of the Ruby gene with the full retroelement insertion (Tcs1 for Tarocco and Doppio Sanguigno and Tcs2 for Jingxian). Band 2 is the AseI fragment at the 5′ end of the Ruby gene containing the solo LTR insertion. Band 3 is the AseI fragment at the 5′ end of the Ruby gene without any insertion (ie the wild type R allele) present in blond oranges. (B) Southern blot of genomic DNA from different blood and blond orange varieties. Genomic DNA was digested with AseI and probed with a ³²P-labelled probe of the Ruby gene from 465 bp upstream of the ATG to the stop codon of the gene. The gel was run for longer than that shown in FIG. 2A, so that only the 5′ fragments of the Ruby gene with insertions remained on the gel. Varieties: T=Tarocco (Reeds, UK); N=Navelina; S=Sanguinelli; MS=Maltaise Sanguine; J=Jingxian; O=Oroval. The presence of DNA containing only the solo LTR (band 2) as well as DNA containing the full Tcs1 retroelement (band 1) is apparent in Sanguinelli and Maltaise Sanguine accessions.

FIG. 6. Maps of structures of the Ruby locus in the different orange accessions and hybrids. Green thick arrows, retrotransposon LTRs; grey thick bars, open reading frame encoding functional Gag-Pol polyprotein; purple thick bars, open reading frame encoding the Ruby protein; thin bars, non-coding regions. The asterisk indicates a stop codon. The differences in sequence between Tcs1 and Tcs2 are indicated by blue lines on the map of Tcs2. The start of transcription of the Ruby mRNA is shown by a grey arrow for each accession. The AseI sites (AseI does not cut in either Tcs1 or Tcs2) in the Ruby locus are shown as vertical line with a circle attached to the upper end. Moro (I) indicates the accession of Moro from CRA, Sicily, and Moro (UK) indicates the accession of Moro from Reeds, UK.

FIG. 7. Expression of Tcs1, Ruby and anthocyanin production in blood and blond oranges in response to cold storage. (A) Detailed map of the Ruby locus with the full Tcs1 retroelement insertion, showing the start of transcription of the Ruby gene. The map of the Ruby transcript is shown above; the thick green bars indicating sequence from the LTR in the 5′UTR, the thick purple bars indicating the open reading frame encoding the Ruby protein, also indicated by thick pink bars in the transcript; the splice sites for the transcript are shown. The positions of the oligonucleotides used for measuring transcript levels of Ruby and Tcs1 are also indicated by colour-coded inverted triangles. (B) Levels of Ruby transcripts in Moro and Tarocco blood orange accessions and in Valencia and Navelina blond orange accessions from CRA in fruit picked fresh and following storage for 30 days at 4° C., determined by qRT-PCR. (C) Levels of Tcs1 transcripts from the LTR in Moro and Tarocco blood orange accessions and in Valencia and Navelina blond orange accessions from CRA in fruit picked fresh and following storage for 30 days at 4° C., determined by qRT-PCR. (D) Levels of Tcs1 and Tcs2 transcripts from the Gag-Pol region of the element in Moro and Tarocco blood orange accessions and in Valencia and Navelina blond orange accessions from CRA in fruit picked fresh and following storage for 30 days at 4° C., determined by qRT-PCR. (E) Levels of anthocyanins in Moro and Tarocco blood orange accessions and in Valencia and Navelina blond orange accessions from CRA in fruit picked fresh and following storage for 30 days at 4° C.

FIG. 8. Southern blots of genomic DNA from Tarocco and Oroval (mandarin) parents and the OTA hybrids. Genomic DNA was digested with AseI and probed with a ³²P-labelled probe of the Tcs1 LTR. AseI does not cut within Tcs1; therefore, fragments larger than 5.4 kb likely represent full length retroelement insertions. (A) Short exposure shows large, strongly hybridising bands (two copies of the LTR). New bands present in the hybrids but not in the parents are arrowed and likely represent new transpositions. (B) Long exposure shows smaller, more weakly hybridizing bands. New bands present in the hybrids but not in the parents are arrowed and likely represent unequal crossing over events between the LTRs to leave solo LTR insertions. T (I)=Tarocco (CRA); O=Oroval; T+O=equimolar mixture of Tarocco and Oroval DNA.

FIG. 9. CsMYC2 regulates anthocyanin biosynthetic gene expression. MYB transcription factors interact with bHLH and WDR proteins in a complex known as the MBW complex. The WDR partner is generally constitutively expressed (Walker et al., 1999) but activity of the bHLH partner can limit anthocyanin production (Hellens et al., 2010; Bradley et al., 1999; Lauter et al., 2004). Transfection assays in tobacco protoplasts using reporter genes driven by the F3H and DFR promoters from Antirrhinum majus were used to assay the ability of different transcription factors to activate the expression of anthocyanin biosynthetic genes. No activation of either promoter was observed with CsMYC2 on its own. The MYB gene Rosea1 activated expression of both promoters slightly on its own. The combination of CsMYC2 with Rosea1 significantly enhanced expression from both the F3H and DFR promoters, showing that CsMYC2 can interact in the MBW complex to activate anthocyanin biosynthesis. Control lanes show GUS activity from protoplasts transfected with the reporter construct alone without added regulatory genes. Data are presented as means (±SD) of three biological replicates.

FIG. 10. Expression analysis of Ruby. Expression analysis of Ruby in varieties of the ‘Sanguigni,’ and ‘Sanguinelli’ blood orange groups. Red pigmentation in the last sample, Vaniglia Sanguigno, is due to the carotenoid lycopene, not anthocyanins.

SEQUENCE LISTING

The nucleotide and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three-letter code for amino acids. The nucleotide sequences follow the standard convention of beginning at the 5′ end of the sequence and proceeding forward (i.e., from left to right in each line) to the 3′ end. Only one strand of each nucleotide sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. The amino acid sequences follow the standard convention of beginning at the amino terminus of the sequence and proceeding forward (i.e., from left to right in each line) to the carboxy terminus. The coding sequences of the present invention as used herein do not include the stop codon unless indicated otherwise or apparent from the context. It is understood that a stop codon can be added at the end of any coding sequence for the termination of transcription. Such stop codons include, for example, TAG, TAA, and TGA.

SEQ ID NO: 1 sets forth a nucleotide sequence of the wild-type allele of the Ruby gene from Citrus sinensis cv. Navel. The coding sequence comprises nucleotides 681-798, 896-1025, and 1821-2358.

SEQ ID NO: 2 sets forth the amino acid sequence of the wild-type Ruby protein of Citrus sinensis cv. Navel, which is encoded by wild-type allele of the Ruby gene set forth in SEQ ID NO: 1.

SEQ ID NO: 3 sets forth a nucleotide sequence of a dominant allele of the Ruby gene from Citrus sinensis cv. Moro. The coding sequence comprises nucleotides 1180-1297, 1395-1524, and 2320-2857.

SEQ ID NO: 4 sets forth the amino acid sequence of the Ruby protein of Citrus sinensis cv. Moro, which is encoded by the dominant allele of the Ruby gene set forth in SEQ ID NO: 3.

SEQ ID NO: 5 sets forth a nucleotide sequence of a deletion allele of the Ruby gene from Citrus sinensis×Citrus reticulata cv. OTA7. The coding sequence comprises nucleotides 836-874.

SEQ ID NO: 6 sets forth the amino acid sequence of the Ruby protein of Citrus sinensis×Citrus reticulata cv. OTA7, which is encoded by the deletion allele of the Ruby gene set forth in SEQ ID NO: 5.

SEQ ID NO: 7 sets forth a nucleotide sequence of the Tcs1 retrotransposon of Citrus sinensis cv. Tarocco. The coding sequence comprises nucleotides 911-4837.

SEQ ID NO: 8 sets forth the amino acid sequence of the polyprotein encoded by the Tcs1 retrotransposon set forth in SEQ ID NO: 7.

SEQ ID NO: 9 sets forth a nucleotide sequence of the Tcs2 retrotransposon of Citrus sinensis cv. Jingxian. The coding sequence comprises nucleotides 964-4890.

SEQ ID NO: 10 sets forth the amino acid sequence of the polyprotein encoded by the Tcs2 retrotransposon set forth in SEQ ID NO: 9.

SEQ ID NO: 11 sets forth a nucleotide sequence of a full-length Ruby mRNA of Citrus sinensis cv. Moro. The coding sequence comprises nucleotides 324-1109.

SEQ ID NO: 12 sets forth the amino acid sequence encoded by the full-length Ruby mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 11.

SEQ ID NO: 13 sets forth a nucleotide sequence of a Ruby mRNA of Citrus sinensis cv. Jingxian. The coding sequence comprises nucleotides 130-915.

SEQ ID NO: 14 sets forth the amino acid sequence encoded by the Ruby mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 13.

SEQ ID NO: 15 sets forth a nucleotide sequence a promoter derived the β-LCY2 gene of Citrus sinensis.

SEQ ID NO: 16 sets forth a nucleotide sequence of Citrus sinensis capsanthin/capsorubin synthase (CCS) gene of GenBank Accession No. AF169241. The coding sequence comprises nucleotides 1722-3230.

SEQ ID NO: 17 sets forth the amino acid sequence of the capsanthin/capsorubin synthase (CCS) encoded by the nucleotide sequence set forth in SEQ ID NO: 16.

SEQ ID NOS: 18-42 are the oligonucleotide primers that are described in Table 1 below.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the inventions are shown. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

The present invention is based on the isolation of the Ruby gene from blood oranges (Citrus sinensis) and the discovery that this gene encodes a novel R2R3 Myb transcription factor that regulates the expression of genes required for anthocyanin biosynthesis in citrus fruit as disclosed in the Example below. It was further discovered that the fruit-specific and cold-dependent accumulation of anthocyanins in blood oranges is the result of retrotransposons that regulate the expression of the Ruby gene in a fruit-specific and cold-dependent manner thereby resulting in the production of the Ruby protein which in turn regulates the expression of genes required for anthocyanin biosynthesis in citrus fruit. Although blond oranges (Citrus sinesis) contain an allele of Ruby that encodes an apparently functional protein, the expression of the Ruby was not detected in blond orange fruit as described below in the Example.

The present invention provides methods for making a citrus plant that is capable of producing fruit with increased levels of anthocyanins in the fruit when compared to a citrus fruit from a wild-type or other control plant. Such methods find use in making citrus trees that are capable of producing fruit with high levels of anthocyanins, whereby anthocyanin production is not cold-dependent as it is for wild-type blood oranges. Such methods find use in the stable production of blood oranges, particularly in the stable production of blood oranges in citrus-growing regions of the world where cold temperatures do not occur or do not reliably occur during fruit development and maturation. Moreover, the methods of the present invention are applicable to other citrus besides oranges. For example, the methods disclosed herein can used to increase the anthocyanin content of the widely consumed, lycopene-rich red grapefruit that is produced from grapefruit varieties such as, for example, ‘Ruby Red’, ‘Henderson’, ‘Ray’, ‘Rio Red’, and ‘Star Ruby’. Thus, the methods disclosed herein find use in making grapefruit that are rich not only in the carotenoid lycopene but also anthocyanins. Such grapefruits, and juices and other food products make therefrom are expected to be highly desirable for human consumption to the presence the high levels of the antioxidants, lycopene and anthocyanins.

The methods of the present invention comprise increasing the expression of Ruby in a citrus plant, particularly in the fruit of a citrus plant. In one embodiment, the methods comprise introducing into at least one citrus plant cell a polynucleotide construct comprising a promoter operably linked to nucleotide sequence encoding Ruby. Preferably, the promoter is capable of driving the expression of the nucleotide sequence encoding Ruby in a citrus fruit or part thereof, particularly the carpels or endocarp. The methods of the invention can further comprise regenerating a citrus plant comprising the polynucleotide construct.

In another embodiment, the methods comprise introducing into at least one citrus plant cell a polynucleotide construct comprising a promoter that is capable of driving the expression of an operably linked nucleotide sequence. In this embodiment, the citrus plant cell comprises stably incorporated in its genome a native or non-native nucleotide sequence encoding a functional Ruby protein. Such a method further comprises the use of homologous recombination methods that are known in the art to incorporate the introduced polynucleotide construct comprising a promoter in operable linkage with the nucleotide sequence encoding a functional Ruby protein, whereby the promoter is capable of driving the expression of the nucleotide sequence encoding a functional Ruby protein. Preferably, the promoter is capable of driving the expression of the nucleotide sequence encoding Ruby in a citrus fruit or part thereof, particularly the carpels or endocarp. The methods of the invention can further comprise regenerating a citrus plant comprising the polynucleotide construct.

Additional embodiments of the invention are described hereinbelow. Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Embodiments of the invention include, but are not limited to:

• • 1. A method for making a plant that is capable of producing a fruit with an increased level of anthocyanins, said method comprising modifying a plant so was to increase the expression of Ruby in the fruit of the plant, wherein a fruit produced by the plant comprises an increased level of anthocyanins when compared to a control fruit.
  • 2. The method of embodiment 1, wherein modifying the plant comprises introducing into at least one cell of the plant a polynucleotide construct comprising a promoter operably linked to a nucleotide sequence encoding Ruby.
  • 3. The method of embodiment 1 or 2, wherein the nucleotide sequence encoding Ruby comprises a nucleotide sequence selected from the group consisting of:
    • (a) the coding sequence set forth in Sequence SEQ ID NO: 1, 3, 11, or 13;
    • (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, or 14;
    • (c) a nucleotide sequence comprising at least 75% identity to at least one of the full-length coding sequences set forth in SEQ ID NOS: 1, 3, 11, and 13, wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;
    • (d) a nucleotide sequence encoding an amino acid sequence comprising at least 75% identity to at least one of the full-length amino acid sequences set forth in SEQ ID NOS: 2, 4, 12, and 14; wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;
    • (e) a fragment of any one of (a)-(d), wherein the fragment encodes a polypeptide comprising Ruby transcription factor activity; and
    • (f) the nucleotide sequence of any one of (a)-(e), wherein the polypeptide further comprises at least one of the motifs, DLX₂RX₃LX₆LX₃R (SEQ ID NO: 49) and KPXPR(S/T)F (SEQ ID NO: 50).
  • 4. The method of embodiment 2 or 3, wherein the promoter preferentially drives gene expression in a fruit.
  • 5. The method of any one of embodiments 2-4, wherein the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 15.
  • 6. The method of any one of embodiments 1-5, wherein the plant is a citrus plant.
  • 7. The method of embodiment 6, wherein the citrus plant is selected from the group consisting of sweet orange, sour orange, grapefruit, pummelo, citron, lime, mandarin, clementine, and lemon.
  • 8. The method of embodiment 6, wherein the citrus plant is a sweet orange.
  • 9. The method of embodiment 8, wherein the sweet orange is a blond orange.
  • 10. The method of embodiment 8, wherein the sweet orange is a blood orange.
  • 11. The method of any one of embodiments 4-10, wherein the promoter drives expression in the carpels of the fruit.
  • 12. The method of any one of embodiments 1-11, further comprising regenerating a plant comprising the polynucleotide construct.
  • 13. The method of embodiment 12, further comprising growing the plant so as to produce at least one fruit.
  • 14. The method of embodiment 13, wherein the fruit produced by the plant comprises an increased level of anthocyanins when compared to a control fruit.
  • 15. The method of any one of embodiments 2-14, wherein the polynucleotide construct is stably incorporated into the genome of the plant.
  • 16. The method of embodiment 1, wherein modifying the plant comprises introducing into at least cell of the plant a polynucleotide construct comprising a promoter that is capable of driving the expression of an operably linked nucleotide sequence in the plant, whereby the polynucleotide construct integrates into the genome of the plant cell in operable linkage with a nucleotide sequence encoding a functional Ruby protein that is present in the genome of plant.
  • 17. The method of embodiment 16, wherein the nucleotide sequence encoding a functional Ruby protein is native to the genome of the plant.
  • 18. The method of embodiment 16 or 17, wherein the nucleotide sequence encoding a functional Ruby protein is heterologous to the genome of the plant.
  • 19. The method of any one of embodiments 16-18, wherein the promoter preferentially drives gene expression in a fruit.
  • 20. The method of any one of embodiments 16-19, wherein the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 15.
  • 21. The method of any one of embodiments 16-20, wherein the plant is a citrus plant.
  • 22. The method of embodiment 21, wherein the citrus plant is selected from the group consisting of sweet orange, sour orange, grapefruit, pummelo, citron, lime, mandarin, clementine, and lemon.
  • 23. The method of embodiment 21, wherein the citrus plant is a sweet orange.
  • 24. The method of embodiment 23, wherein the sweet orange is a blond orange.
  • 25. The method of embodiment 23, wherein the sweet orange is a blood orange.
  • 26. The method of any one of embodiments 19-25, wherein the promoter drives expression in the carpels of the fruit.
  • 27. The method of any one of embodiments 16-26, further comprising regenerating a plant comprising the polynucleotide construct.
  • 28. The method of embodiment 27, further comprising growing the plant so as to produce at least one fruit.
  • 29. The method of embodiment 30, wherein the fruit produced by the plant comprises an increased level of anthocyanins when compared to a control fruit.
  • 30. The method of embodiment 16, wherein the polynucleotide construct is stably incorporated into the genome of the plant.
  • 31. A plant produced by the method of any one of embodiments 1-30.
  • 32. A scion or other part or cell of the plant of embodiment 31.
  • 33. A fruit produced by the plant of embodiment 31.
  • 34. A juice or food product produced from the fruit of embodiment 33.
  • 35. A transformed plant or plant cell comprising stably incorporated in its genome a polynucleotide construct, said polynucleotide construct comprising a promoter operably linked to a nucleotide sequence, wherein said nucleotide sequence comprises a member selected from the group consisting of:
    • (a) the coding sequence set forth in SEQ ID NO: 1, 3, 11, or 13;
    • (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, or 14;
    • (c) a nucleotide sequence comprising at least 75% identity to at least one of the full-length coding sequences set forth in SEQ ID NOS: 1, 3, 11, and 13, wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;
    • (d) a nucleotide sequence encoding an amino acid sequence comprising at least 75% identity to at least one of the full-length amino acid sequences set forth in SEQ ID NOS: 2, 4, 12, and 14; wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;
    • (e) a fragment of any one of (a)-(d), wherein the fragment encodes a polypeptide comprising Ruby transcription factor activity;
    • (f) the nucleotide sequence of any one of (a)-(e), wherein the polypeptide further comprises at least one of the motifs, DLX₂RX₃LX₆LX₃R (SEQ ID NO: 49) and KPXPR(S/T)F (SEQ ID NO: 50); and (g) a nucleotide sequence that is fully complementary to any one of (a)-(f).
  • 36. The plant or plant cell of embodiment 35, wherein the plant is a citrus plant.
  • 37. The plant or plant cell of embodiment 36, wherein the citrus plant is selected from the group consisting of sweet orange, sour orange, grapefruit, pummelo, citron, lime, mandarin, clementine, and lemon.
  • 38. The plant or plant cell of embodiment 36, wherein the citrus plant is a sweet orange.
  • 39. The plant or plant cell of embodiment 38, wherein the sweet orange is a blond orange.
  • 40. The plant or plant cell of embodiment 38, wherein the sweet orange is a blood orange.
  • 41. The plant or plant cell of any one of embodiments 35-40, wherein the promoter preferentially drives gene expression in a fruit.
  • 42. The plant or plant cell of any one of embodiments 35-40, wherein the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 15.
  • 43. The plant or plant cell of embodiment 41 or 42, wherein the promoter drives expression in the carpels of the fruit.
  • 44. The plant of any one of embodiments 36-43, wherein a fruit produced by the plant comprises an increased level of anthocyanins when compared to a control fruit.
  • 45. The plant cell of any one of embodiments 36-43, wherein the plant cell is a fruit cell.
  • 46. A fruit produced by the citrus plant of any one or embodiments 36-44.
  • 47. A transformed plant or plant cell comprising stably incorporated in its genome a polynucleotide construct, said polynucleotide construct comprising a promoter, wherein the polynucleotide construct is in operable linkage with a nucleotide sequence encoding a functional Ruby protein that is present in the genome of plant.
  • 48. The transformed plant or plant cell of embodiment 47, wherein Ruby expression is increased in the plant or at least one part thereof relative to a control plant.
  • 49. The transformed plant or plant cell of embodiment 47 or 48, wherein the nucleotide sequence encoding a functional Ruby protein is native to the genome of the plant.
  • 50. The transformed plant or plant cell of embodiment 47 or 48, wherein the nucleotide sequence encoding a functional Ruby protein is heterologous to the genome of the plant.
  • 51. The plant or plant cell of any one of embodiments 47-50, wherein the plant is a citrus plant.
  • 52. The plant or plant cell of embodiment 51, wherein the citrus plant is selected from the group consisting of sweet orange, sour orange, grapefruit, pummelo, citron, lime, mandarin, clementine, and lemon.
  • 53. The plant or plant cell of embodiment 51, wherein the citrus plant is a sweet orange.
  • 54. The plant or plant cell of embodiment 53, wherein the sweet orange is a blond orange.
  • 55. The plant or plant cell of embodiment 53, wherein the sweet orange is a blood orange.
  • 56. The plant or plant cell of any one of embodiments 47-55, wherein the promoter preferentially drives gene expression in a fruit.
  • 57. The plant or plant cell of any one of embodiments 47-56, wherein the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 15.
  • 58. The plant or plant cell of embodiment 56 or 57, wherein the promoter drives expression in the carpels of the fruit.
  • 59. The plant of any one of embodiments 47-58, wherein a fruit produced by the plant comprises an increased level of anthocyanins when compared to a control fruit.
  • 60. The plant cell of any one of embodiments 47-58, wherein the plant cell is a fruit cell.
  • 61. A fruit produced by the citrus plant of any one or embodiments 47-59.
  • 62. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
    • (a) the coding sequence set forth in SEQ ID NO: 1, 3, 11, or 13;
    • (b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, or 14;
    • (c) a nucleotide sequence comprising at least 75% identity to at least one of the full-length coding sequences set forth in SEQ ID NOS: 1, 3, 11, and 13, wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;
    • (d) a nucleotide sequence encoding an amino acid sequence comprising at least 75% identity to at least one of the full-length amino acid sequences set forth in SEQ ID NOS: 2, 4, 12, and 14; wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;
    • (e) a fragment of any one of (a)-(d), wherein the fragment encodes a polypeptide comprising Ruby transcription factor activity;
    • (f) the nucleotide sequence of any one of (a)-(e), wherein the polypeptide further comprises at least one of the motifs, DLX₂RX₃LX₆LX₃R (SEQ ID NO: 49) and KPXPR(S/T)F (SEQ ID NO: 50); and
    • (g) a nucleotide sequence that is fully complementary to any one of (a)-(f).
  • 63. An expression cassette comprising a promoter operably linked to the nucleic acid molecule of embodiment 62.
  • 64. A host cell comprising the expression cassette of embodiment 63.
  • 65. The host cell of embodiment 64, wherein the host cell is a plant cell.
  • 66. A plant comprising the expression cassette of embodiment 63.
  • 67. A fruit, a fruit juice, or other food product comprising the expression cassette of embodiment 63.
  • 68. A polypeptide comprising an amino acid sequence selected from the group consisting of:
    • (a) the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, or 14;
    • (b) an amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1, 3, 11, or 13;
    • (c) an amino acid sequence comprising at least 75% identity to at least one of the full-length amino acid sequences set forth in SEQ ID NOS: 2, 4, 12, and 14, wherein the polypeptide comprises Ruby transcription factor activity;
    • (d) an amino acid sequence comprising a nucleotide sequence comprising at least 75% identity to at least one of the full-length coding sequences set forth in SEQ ID NOS: 1, 3, 11, and 13, wherein the polypeptide comprises Ruby transcription factor activity; and
    • (e) a fragment of any one of (a)-(d), wherein the polypeptide comprises Ruby transcription factor activity.
  • 69. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
    • (a) the nucleotide sequence set forth in SEQ ID NO: 7 or 9;
    • (b) the coding sequence set forth in SEQ ID NO: 7 or 9;
    • (c) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 8 or 10;
    • (d) a functional variant or fragment of the nucleotide sequence of (a), wherein the functional fragment or variant comprise at least 75% identity to at least one of the full-length nucleotide sequences set forth in SEQ ID NOS: 7 and 8;
    • (e) a nucleotide sequence encoding a functional fragment or variant of the protein encoding by (b) or (c), wherein the functional fragment or variant comprises at least 75% identity to at least one of the full-length amino acid sequences set forth in SEQ ID NOS: 8 and 10; and
    • (f) a nucleotide sequence that is fully complementary to any one of (a)-(e).
  • 70. A plant, plant cell, or expression cassette comprising the nucleic acid of embodiment 69.
  • 71. A polypeptide comprising an amino acid sequence selected from the group consisting of:
    • (a) the amino acid sequence set forth in SEQ ID NO: 8; and
    • (b) the amino acid sequence set forth in SEQ ID NO: 9.

Isolated nucleic acid molecules comprising the Ruby nucleotide sequences disclosed herein and fragments and variants thereof that encode functional Ruby proteins are further provided as wells as the Ruby proteins encoded thereby. Also provided are nucleic acid molecules comprising the retrotransposons, Tcs1 and Tcs2, set forth in SEQ ID NOS: 7 and 9, respectively, and fragments and variants thereof that encode functional retrotransposons. Such nucleic acid molecule acid molecules find use in methods of transposon tagging or for regulating gene expression in citrus in a fruit-specific, cold-dependent manner as disclosed hereinbelow. Expression cassettes comprising a promoter operably linked to a nucleotide sequence encoding a Ruby protein and plants, plant parts, plant cells and other host cells comprising a nucleotide sequence encoding a Ruby protein are also provided. Further provided are plants, plant parts, plant cells, and other host cells comprising the nucleotide sequence or Tcs1 and/or Tcs2 and fragments and variants thereof that encode functional retrotransposons.

Additionally provided are citrus plants and citrus plant cell made by the methods disclosed herein as well as citrus fruit produced from such plants and food products derived from the citrus fruit including, for example, citrus fruit juice, beverages comprising citrus fruit juice (e.g., sodas, smoothies and other blended beverages). marmalades, and food colorants.

The invention encompasses isolated or substantially purified polynucleotide (also referred to herein as “nucleic acid molecules”) or protein (also referred to herein as “polypeptide”) compositions. An “isolated” or “purified” polynucleotide or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an “isolated” polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By “fragment” is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of polynucleotides comprising coding sequences may encode protein fragments that retain biological activity of the native protein and hence R2R3 Myb transcription factor as disclosed herein below for Ruby. Fragments of polynucleotide comprising retrotransposon sequences retain biological activity of the native the native Tcs1 or Tcs2 retrotransposon. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the invention.

A fragment of a Ruby polynucleotide that encodes a biologically active portion of a Ruby protein of the invention will encode at least 15, 25, 30, 50, 100, 150, 200, or 250 contiguous amino acids, or up to the total number of amino acids present in a full-length Ruby protein of the invention (for example, 262 amino acids for the amino acid sequences set forth in SEQ ID NOS: 2, 4, 12, and 14). Fragments of a Ruby polynucleotide that are useful as hybridization probes or PCR primers generally need not encode a biologically active portion of a Ruby protein.

Thus, a fragment of a Ruby polynucleotide may encode a biologically active portion of a Ruby protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of a Ruby protein can be prepared by isolating a portion of one of the Ruby polynucleotides of the invention, expressing the encoded portion of the Ruby protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the Ruby protein. Polynucleotides that are fragments of a Ruby nucleotide sequence comprise at least 16, 20, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or 750 contiguous nucleotides, or up to the number of nucleotides present in a full-length Ruby polynucleotide disclosed herein (for example, 786 nucleotides for the coding sequence set forth in SEQ ID NO: 11; the coding sequence of SEQ ID NO: 11 comprises nucleotides 324 to 1109 with the stop codon immediately thereafter at nucleotides 1110-1112).

“Variants” is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the Ruby polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a Ruby protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.

Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, an isolated polynucleotide that encodes a polypeptide with a given percent sequence identity to the Ruby polypeptide the Ruby comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

“Variant” protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, Ruby transcription factor activity as described in the Example hereinbelow. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native Ruby protein of the invention will have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

To assess if variants and fragments of Ruby are functional or active, Ruby transcription factor activity can be assessed by determining if a variant or fragment protein can activate anthocyanin biosynthesis under the control of the constitutive CaMV 35S promoter in tobacco by the method disclosed in the Example hereinbelow.

The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be optimal.

Thus, the genes and polynucleotides of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired biological activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and optimally will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity of a Ruby protein be can be evaluated by the assay as described hereinbelow. Those fragments and variants of an Ruby protein will retain the ability of Ruby to activate anthocyanin biosynthesis in a plant or plant cell and thus, comprise Ruby transcription factor activity.

Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

The polynucleotides of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. “Orthologs” is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated polynucleotides that have promoter activity and which hybridize under stringent conditions to at least one of the polynucleotides disclosed herein, or to variants or fragments thereof, are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other corresponding polynucleotides present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as ³²P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the polynucleotides of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

For example, an entire nucleic acid molecule of polynucleotide disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding polynucleotide and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among one or more of the polynucleotide sequences of the present invention and are optimally at least about 10 nucleotides in length, and most optimally at least about 20 nucleotides in length. Such probes may be used to amplify corresponding polynucleotides from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, optimally less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours. The duration of the wash time will be at least a length of time sufficient to reach equilibrium.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T_m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T_m=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T_m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T_m is reduced by about 1° C. for each 1% of mismatching; thus, T_m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the T_m can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T_m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T_m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T_m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (T_m). Using the equation, hybridization and wash compositions, and desired T_m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T_m of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is optimal to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

It is recognized that the polynucleotide molecules of the present invention encompass polynucleotide molecules comprising a nucleotide sequence that is sufficiently identical to one of the nucleotide sequences set forth in SEQ ID NOS: 6, 7, 9, 11, 13-18, 20, 22, or 24. The term “sufficiently identical” is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent nucleotides to a second nucleotide sequence such that the first and second nucleotide sequences have a common structural domain and/or common functional activity. For example, nucleotide sequences that contain a common structural domain having at least about 45%, 55%, or 65% identity, preferably 75% identity, more preferably 85%, 90%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently identical.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the polynucleotide molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (available on the World Wide Web at www.ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the full-length sequences of the invention and using multiple alignment by mean of the algorithm Clustal W (Nucleic Acid Research, 22(22):4673-4680, 1994) using the program AlignX included in the software package Vector NTI Suite Version 7 (InforMax, Inc., Bethesda, Md., USA) using the default parameters; or any equivalent program thereof. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by CLUSTALW (Version 1.83) using default parameters (available at the European Bioinformatics website on the World Wide Web at ebi.ac.uk/Tools/clustalw/index).

The use of the term “polynucleotide” is not intended to limit the present invention to polynucleotides comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides, can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

The Ruby polynucleotides of the invention comprising Ruby protein coding sequences can be provided in expression cassettes for expression in the plant or other organism or non-human host cell of interest. The cassette will include 5′ and 3′ regulatory sequences operably linked to a Ruby polynucleotide of the invention. “Operably linked” is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide or gene of interest and a regulatory sequence (i.e., a promoter) is functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the Ruby polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

The expression cassette will include in the 5′-3′ direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), polynucleotide to be expressed, and a transcriptional and translational termination region (i.e., termination region) functional in plants or other organism or host cell. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the polynucleotide to be expressed may be native/analogous to the host cell or to each other. Alternatively, any of the regulatory regions and/or the polynucleotide to be expressed may be heterologous or non-native to the host cell or to each other. As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.

For expression of the Ruby protein in a plant or plant cells, the methods of the invention comprise introducing a polynucleotide construct into a plant comprising a promoter that drives expression in a plant or part or cell thereof. Any promoter known in the art can be used in the methods of the invention including, but not limited to, the tissue-preferred promoters, fruit-preferred promoters, chemical-regulated promoters, and the like. Preferred promoters of the invention are promoters that drive expression in fruit or part thereof. For expression of Ruby in citrus fruit, preferred promoters of the invention are promoters that drive expression in fruit or part thereof, particularly the carpels or endocarp. An example of a preferred promoter of the present invention is set forth in the drawing labeled as SEQ ID NO: 15. Another promoter that can be used in the methods disclosed herein is the promoter of the Citrus sinensis capsanthin/capsorubin synthase (CCS) gene set forth in the drawing labeled as SEQ ID NO: 16.

The termination region may be native with the transcriptional initiation region, may be native with the operably linked polynucleotide of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the polynucleotide of interest, the plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the polynucleotides can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

The expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters for expression in plants. Such constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to target enhanced Ruby expression within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified, if necessary, for weak expression.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14(2):247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al. (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference.

The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present invention.

Numerous plant transformation vectors and methods for transforming plants are available. See, for example, An, G. et al. (1986) Plant Pysiol., 81:301-305; Fry, J., et al. (1987) Plant Cell Rep. 6:321-325; Block, M. (1988) Theor. Appl Genet. 76:767-774; Hinchee, et al. (1990) Stadler. Genet. Symp. 203212.203-212; Cousins, et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, P. P. and Slightom, J. L. (1992) Gene. 118:255-260; Christou, et al. (1992) Trends. Biotechnol. 10:239-246; D'Halluin, et al. (1992) Bio/Technol. 10:309-314; Dhir, et al. (1992) Plant Physiol. 99:81-88; Casas et al. (1993) Proc. Nat. Acad Sci. USA 90:11212-11216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P: 119-124; Davies, et al. (1993) Plant Cell Rep. 12:180-183; Dong, J. A. and Mchughen, A. (1993) Plant Sci. 91:139-148; Franklin, C. I. and Trieu, T. N. (1993) Plant. Physiol. 102:167; Golovkin, et al. (1993) Plant Sci. 90:41-52; Guo Chin Sci. Bull. 38:2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres N. M. and Park, W. D. (1994) Crit. Rev. Plant. Sci. 13:219-239; Barcelo, et al. (1994) Plant. J. 5:583-592; Becker, et al. (1994) Plant. J. 5:299-307; Borkowska et al. (1994) Acta. Physiol Plant. 16:225-230; Christou, P. (1994) Agro. Food. Ind. Hi Tech. 5: 17-27; Eapen et al. (1994) Plant Cell Rep. 13:582-586; Hartman, et al. (1994) Bio-Technology 12: 919923; Ritala, et al. (1994) Plant. Mol. Biol. 24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol. 104:3748.

The methods of the invention involve introducing a polynucleotide construct into a plant. By “introducing” what is intended is presenting to the plant the polynucleotide construct in such a manner that the construct gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a polynucleotide construct to a plant, only that the polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide constructs into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

By “stable transformation” is intended that the polynucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by progeny thereof. By “transient transformation” is intended that a polynucleotide construct introduced into a plant does not integrate into the genome of the plant. In preferred embodiments of the invention, the plants are plants that are stably transformed with a polynucleotide construct of the invention.

Certain embodiments of the methods of the invention involve stably transforming a plant or cell thereof with a polynucleotide construct comprising a promoter operably linked to a Ruby coding sequence. The present invention is not limited to introducing the polynucleotide construct into the plant or plant cell as a single nucleic acid molecule but also includes, for example, introducing two or more nucleic acid molecules that comprise portions of the polynucleotide construct into the plant or plant cell, wherein the two or more nucleic acid collectively comprise the polynucleotide construct. It is recognized that the two or more nucleic acid molecules can be recombined into the polynucleotide construct within a plant cell via homologous recombination methods that are known in the art.

Alternatively, the two or more nucleic acid molecules that comprise portions of the polynucleotide construct can be introduced a plant or cell thereof in a sequential manner. For example, a first nucleic acid molecule comprising a first portion of a polynucleotide construct can be introduced into a plant cell, and the transformed plant cell can then be regenerated into a plant comprising the first nucleic acid molecule. A second nucleic acid molecule comprising a second portion of a polynucleotide construct can then be introduced into a plant cell comprising the first nucleic acid molecule, wherein the first and second nucleic acid molecules are recombined into the polynucleotide construct via homologous recombination methods.

Methods of homologous recombination involve inducing double breaks in DNA using zinc-finger nucleases or homing endonucleases that have been engineered to make double-strand breaks at specific recognition sequences in the genome of a plant, other organism, or host cell. See, for example, Durai et al., (2005) Nucleic Acids Res 33:5978-90; Mani et al. (2005) Biochem Biophys Res Comm 335:447-57; U.S. Pat. Nos. 7,163,824, 7,001,768, and 6,453,242; Arnould et al. (2006) J Mol Biol 355:443-58; Ashworth et al., (2006) Nature 441:656-9; Doyon et al. (2006) J Am Chem Soc 128:2477-84; Rosen et al., (2006) Nucleic Acids Res 34:4791-800; and Smith et al., (2006) Nucleic Acids Res 34:e149; U.S. Pat. App. Pub. No. 2009/0133152; and U.S. Pat. App. Pub. No. 2007/0117128; all of which are herein incorporated in their entirety by reference.

TAL effector nucleases can also be used to make double-strand breaks at specific recognition sequences in the genome of a plant for gene modification or gene replacement through homologous recombination. TAL effector nucleases are a new class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism. TAL effector nucleases are created by fusing a native or engineered TAL effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, FokI. The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. See, WO 2010/079430; Morbitzer et al. (2010) PNAS 10.1073/pnas. 1013133107; Scholze & Boch (2010) Virulence 1:428-432; Christian et al. Genetics (2010) 186:757-761; Li et al. (2010) Nuc. Acids Res. (2010) doi:10.1093/nar/gkq704; and Miller et al. (2011) Nature Biotechnology 29:143-148; all of which are herein incorporated by reference.

In certain embodiments of the invention, such methods of homologous recombination can be used to insert a promoter that is introduced into a plant cell at a position that is in the vicinity of and linked to a native or non-native Ruby coding sequence that is in the genome of the plant cell, whereby the inserted promoter is in operably linkage with the Ruby coding sequence and can drive the expression of the Ruby coding sequence in the plant cell or a plant regenerated therefrom or in any part or parts of the regenerated plant. A preferred plant part for the expression of Ruby is a fruit or cell or tissue thereof. In some embodiments of the invention, the preferred plant is a citrus plant and the preferred plant part is the fruit or the carpels therein. It is recognized that the endocarp of a citrus fruit is comprised of multiple carpels and that it is the carpels which comprise the source of the juice that can be extracted from citrus fruit.

For the transformation of plants and plant cells, the nucleotide sequences of the invention are inserted using standard techniques into any vector known in the art that is suitable for expression of the nucleotide sequences in a plant or plant cell. The selection of the vector depends on the preferred transformation technique and the target plant species to be transformed.

Methodologies for constructing plant expression cassettes and introducing foreign nucleic acids into plants are generally known in the art and have been previously described. For example, foreign DNA can be introduced into plants, using tumor-inducing (Ti) plasmid vectors. Other methods utilized for foreign DNA delivery involve the use of PEG mediated protoplast transformation, electroporation, microinjection whiskers, and biolistics or microprojectile bombardment for direct DNA uptake. Such methods are known in the art. (U.S. Pat. No. 5,405,765 to Vasil et al.; Bilang et al. (1991) Gene 100: 247-250; Scheid et al., (1991) Mol. Gen. Genet., 228: 104-112; Guerche et al., (1987) Plant Science 52: 111-116; Neuhause et al., (1987) Theor. Appl Genet. 75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208:1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlock et al., (1989) Plant Physiology 91: 694-701; Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989). The method of transformation depends upon the plant cell to be transformed, stability of vectors used, expression level of gene products and other parameters.

Other suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection as Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described by Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediated transformation as described by Townsend et al., U.S. Pat. No. 5,563,055, Zhao et al., U.S. Pat. No. 5,981,840, direct gene transfer as described by Paszkowski et al. (1984) EMBO J. 3:2717-2722, and ballistic particle acceleration as described in, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lec1 transformation (WO 00/28058). Also see, Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

The polynucleotides of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that the a protein of the invention may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotide constructs into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931; herein incorporated by reference.

In specific embodiments, the nucleotide sequences of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the nucleotide sequence or variants and fragments thereof directly into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202:179-185; Nomura et al. (1986) Plant Sci. 44:53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107:775-784, all of which are herein incorporated by reference. Alternatively, the nucleotide sequence can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and Agrobacterium tumefaciens-mediated transient expression as described below.

The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present invention provides transformed seed (also referred to as “transgenic seed”) having a polynucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated into their genome.

The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, peppers (Capsicum spp; e.g., Capsicum anmuum, C. baccatum, C. chinense, C. frutescens, C. pubescens, and the like), tomatoes (Lycopersicon esculentum), tobacco (Nicotiana tabacum), eggplant (Solanum melongena), petunia (Petunia spp., e.g., Petunia×hybrida or Petunia hybrida), corn or maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers. Preferred plant species are all Citrus spp. including, but are not limited to, cultivated citrus species, such as, for example, orange, lemon, meyer lemon, lime, key lime, Australian limes, grapefruit, mandarin orange, clementine, tangelo, tangerine, kumquat, pomelo, ugli, sweet orange, blond orange, blood orange, citron, Buddha's hand, and bitter orange. Preferred citrus species are sweet orange (including, for example, blond orange and blood orange), sour orange, grapefruit, pummelo, citron, lime, mandarin, clementine, and lemon.

As used herein, the term “plant” includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, cotyledons, flowers, stems, shoots, hypocotyls, epicotyls, branches, fruits, roots, root tips, buds, anthers, scions, rootstocks, and the like. The present invention encompasses all plants derived from the regenerated plants of invention provided that these derived plants comprise the introduced polynucleotides. Such derived plants can also be referred to herein as derivative plants or derivatives. The term “plant” also encompasses a tree.

The derivative plants or derivatives include, for example, sexually and asexually produced progeny, variants, mutants, and other derivatives of the regenerated plants that comprise at least one of the polynucleotides of the present invention. Also within the scope of the present invention are vegetatively propagated plants including, for example, plants regenerated by cell or tissue culture methods from plant cells, plants tissues, plant organs, other plant parts, or seeds, plants produced by rooting a stem cutting, and plants produced by grafting a scion (e.g., a stem or part thereof, or a bud) onto a rootstock which is the same species as the scion or a different species. Such vegetatively propagated plants or at least one part thereof comprise at least one polynucleotide of the present invention. It is recognized that vegetatively propagated plants are also known as clonally propagated plants, asexually propagated, or asexually reproduced plants.

A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e. with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed. As used herein a control fruit is a fruit produced by a control plant. Similarly, a wild-type fruit is a fruit produced by a wild-type plant.

The invention provides host cells comprising at least one polynucleotide construct or nucleic acid molecule of the present invention. Such host cells include, for example, bacterial cells, fungal cells, animal cells, and plant cells. Preferably, the host cells are non-human, host cells. More preferably, the host cells are plant cells. Most preferably, the host cells are citrus plant cells. Additionally, the invention encompasses viruses and viroids comprising at least one polynucleotide construct or nucleic acid molecule of the present invention.

The present invention provides fruit, particularly citrus fruit, with increased level of anthocyanins when compared to a wild-type fruit. It is recognized that the anthocyanins are water-soluble vacuolar pigments that may appear red, purple, or blue according to the pH, which belong to a parent class of molecules called flavonoids synthesized via the phenylpropanoid pathway. Anthocyanins are known to occur in the tissues of higher plants. Anthocyanins are derivatives of anthocyanidins, which include pendant sugars. As used herein, a fruit has an increased level of anthocyanins when the fruit has an increased level of at least one anthocyanin and/or anthocyanin derivative thereof, when compared to a wild-type or control fruit. In preferred embodiments, a fruit of the invention fruit has an increased level of total anthocyanins when compared to a wild-type or control fruit. In some embodiments, total anthocyanin content in a fruit is the total content of anthocyanins and anthocyanin derivatives in the fruit. In other embodiments, total anthocyanin content in a fruit is the total content of anthocyanins in the fruit exclusive of anthocyanin derivatives.

To further increase the anthocyanin content of the fruit, the method of the present invention comprise increasing the expression of other genes that are known in the art to be involved in the biosynthesis of anthocyanins in a plant, particularly in a fruit. The expression of such genes can be can be modified as disclosed herein for Ruby. An example of one such gene is CsMYC2.

The following examples are offered by way of illustration and not by way of limitation.

Example

Retrotransposons Control Fruit-Specific, Cold-Dependent Accumulation of Anthocyanins in Blood Oranges

Traditionally, Sicilian blood oranges have been associated with cardiovascular health and consumption has been shown to prevent obesity in mice fed a high fat diet. Despite increasing consumer interest in these health-promoting attributes, production of blood oranges remains unreliable, due largely to a dependency on cold for full color formation. We show that Sicilian blood orange arose by insertion of a Copia-like retrotransposon, which controls the expression of an adjacent gene encoding a transcriptional activator of anthocyanin production. Cold dependency reflects the induction of the retroelement by stress. A blood orange of Chinese origin results from an independent insertion of a similar retrotransposon and color formation in its fruit is also cold-dependent. Our results suggest that transposition and recombination of retroelements are likely major sources of variation in Citrus.

Materials and Methods

Plant Material

Citrus sinensis L. Osbeck cv. Moro, Tarocco, Doppio Sanguigno, Cadenera, Navelina and Valencia were grown at the CRA-ACM experimental farm (Palazzelli, Sicily, Italy). Different accessions of Moro, Tarocco and Navelina and the varieties Maltaise Sanguine and Sanguinelli (Spanish) were obtained from the UK National Citrus collection, (Reeds Nursery, Loddon, UK). Fruit and leaves of Citrus sinensis L. Osbeck cv. Jingxian were obtained from Jingzhou, Huannan Province, China. OTA and OMO hybrids grown in Palazzelli were obtained by conventional Citrus breeding methods using controlled pollinations between Oroval mandarin (C. clementina), used as the female parent, and Tarocco 57-1E-1 or Moro NL 58-8D-1 (C. sinensis L. Osbeck), used as the male parent.

Isolation of Ruby cDNA

Total RNA was extracted from Moro (CRA, Sicily) fruit flesh and reverse transcribed using a T7-oligo5 dT primer and SuperScript III RT (Invitrogen). First-strand cDNA was amplified by PCR using degenerate primers BUT-F3 and BUT-R3. The full-length cDNA was isolated using 5′ and 3′ SMART RACE Amplification Kit (Clontech) and gene-specific primers PMC-F1 and PMC-R1. For Jingxian, total RNA was extracted from juice and reverse transcribed using the gene-specific primer PMC-Z and SuperScript III RT. Ruby cDNA was obtained using a 5′ RACE kit (Invitrogen) and gene-specific primers PMC-R1 and LeLe-R1. Primer sequences are provided in Table 1.

TABLE 1
Primers
Name	Sequence	Description
BUT-F3	GGRKTKAGRAARGGTDCATGGAC (SEQ ID NO: 18)	Degenerate primers
BUT-R3	CCARWARTTYTTSACATCRTTWGC (SEQ ID NO: 19)	to
		isolate Ruby partial
		cDNA
PMC-R1	TTCCCGGAAGCCTGCCCACAATCA(SEQ ID NO: 20)	Ruby 5′ and 3′
PMC-F1	CATGGACAGGAGAGGAAGATGATCT (SEQ ID NO: 21)	RACE
		from Moro flesh
		cDNA
PMC-Z	CTTACTTTGCATTGAGAAGATCCCA (SEQ ID NO: 22)	Ruby 5′ and 3′
PMC-R1	TTCCCGGAAGCCTGCCCACAATCA (SEQ ID NO: 23)	RACE
		from Jingxian juice
LeLe-R1	CAACTTCATCTGCTGCAAATTCTCCT (SEQ ID NO: 24)	cDNA
PMC-GWF	GGGGACAAGTTTGTACAAAAAAGCAGGCTATGGCGGATTCCTTAGGAGTT	Expression of Ruby
	(SEQ ID NO: 25)	in
PMC-GWR	GGGACCACTTTGTACAAGAAAGCTGGGTCTTACTTTGCATTGAGAAGATC	tobacco
	(SEQ ID NO: 26)
PMC-47	TCCTCTCCTGTCCATGCACCTTTACGAAC (SEQ ID NO: 27)	Chromosome
PMC-109	GAGGAACTTGATGCCATTTTGCTTCCCCA (SEQ ID NO: 28)	walking for
		Isolation of Ruby
		promoters
PMC-CF	CATTGAAGCAGGCCAGAGTTGTCCGACTGATGAC (SEQ ID NO: 29)	iPCR for isolation of
PMC-NR1	CTCTCCTGTCCATGCACCTTTACGAACTCCTAAG (SEQ ID NO: 30)	Tcs1
PMC-G3	GAGAGTATACCGTATGCGTACACA (SEQ ID NO: 31)	Isolation of full-
PMC-U	ACACGTAGCTATTGGACCACCCT (SEQ ID NO: 32)	length Tcs1
PMCi4	GCCGAAAAGTCTCCAGTAGTGACAAAGGTGACAG (SEQ ID NO: 33)	iPCR for isolation of
PMCiD	TCGCTGTTCTTCCCGGAAGCCTGCCCACAATCAG (SEQ ID NO: 34)	deletion allele and
		Tcs2
PMC-G2	CCGAAATACAGAATGCTCAAATGGGA (SEQ ID NO: 35)	Isolation of full-
PMC-S5	AGCACCTACACTTACTCAACTCTC (SEQ ID NO: 36)	length Tcs2
PMC2ES-Fw	AGCTGCTGGGCAACAGATGGT (SEQ ID NO: 37)	Ruby expression
PMC2ES-Rev	CTTCACATCGTTCGCTGTTC (SEQ ID NO: 38)	qRT-PCR
GAL-POL1-Fw	GCCCGTGGACGTAGGCTAA (SEQ ID NO: 39)	Tcs1 LTR expression
GAL-POL1-Rev	AAGAACAAGCACAAAAGAAAATACCA (SEQ ID NO: 40)	qRT-PCR
GAL-POL4-Fw	TGACAGTCAGAGTGCCTTGCA (SEQ ID NO: 41)	Tcs1 Gag-Pol
GAL-POL4-Rev	TCCTATGTGCTTTGTCCTGGAA (SEQ ID NO: 42)	expression
		qRT-PCR

Phylogenetic Analysis

Protein sequences from Arabidopsis and selected Myb proteins from other species belonging to subgroups 2, 4, 5, 6 and 7 were aligned using PRANK (Loytynoja and Goldman, 2008). The alignment of the DNA binding domain only was used to calculate distance estimates (the Jones Taylor Thornton matrix (JTT) model of evolution) for a neighbour-joining tree with the PHYLIP software package (Felsenstein et al, 1994). To provide statistical support for each node in the tree, a consensus tree was generated from 1000 bootstrap data sets.

Ectopic Expression of Ruby in Tobacco

The coding sequence of the Ruby cDNA was amplified with primers PMC-GWF and PMC-GWR and cloned in a pBin19-derived binary vector, previously equipped with a double CaMV 35S promoter, the CaMV Terminator with attR recombination sites in between, using Gateway® recombination technology (Invitrogen). The resulting plasmid was transferred to Agrobacterium tumefaciens strain LBA4404 and used to transform Nicotiana tabacum (cv. Samsun).

Protoplast Transfection Assays

Tobacco protoplasts were isolated from 3-5 week old leaves of Nicotiana Tabacum cv Samsun following the procedure described by Negrutiu et al. (Negrutiu et al., 1987). For each transfection, 10 μg of plasmid DNA containing the flavanone-3-hydroxylase (F3H) or dihydroflavonol-4-reductase (DFR) promoters from Antirrhinum majus fused to the 3-glucuronidase (GUS) reporter gene were used. To measure expression from the promoters, plasmid DNA containing the cDNA sequences coding for the transcriptional activators AmRosea1 (4 μg: Schwinn et al., 2006) and CsMYC2 (5.5 μg) under the control of the double CaMV35 S promoter were used. Different amounts of an empty plasmid containing the double CaMV35S promoter were used to ensure that equal amounts of total DNA and viral promoter were used for each transfection. After incubation for 40 h, protoplasts were collected by centrifugation and GUS activity in the cell lysate was determined according to Jefferson (Jefferson, 1987) and expressed as nmol methylumbelliferone per mg protein per minute. All transfections were performed in triplicate and GUS activity was measured in duplicate for each transfection.

Southern Blots

Citrus leaves were ground in liquid nitrogen and DNA was extracted using caesium chloride density gradient purification. DNA (10 □g per sample) was digested with AseI (and numerous other restriction enzymes for mapping) for 5 h and then separated by electrophoresis. Denatured DNA was transferred to nitrocellulose membrane filters. Filters were hybridised with ³²P-labelled probes overnight at 60° C. and washed in 0.1×SSC, 0.5% SDS at 60° C. for 2 h before exposure to X-Ray film (Fuji RX-100).

Isolation of Ruby Promoters

The upstream regions of Ruby from Moro and Cadenera (CRA, Sicily) were isolated by chromosome walking using the GenomeWalker Kit (Clontech) and gene-specific primers PMC-47 and PMC-109.

Isolation of Tcs1, Tcs2 and Ruby Deletion Allele

A Tcs1 fragment was initially obtained from Tarocco and Moro (Reeds, UK) DNA from leaves was digested with BsrGI and self-ligated. The Tcs1 fragment was isolated by inverse PCR using primers PMC-CF and PMC-NR1. Full-length Tcs1 was obtained by conventional PCR using primers PMC-G3 and PMC-U. A Tcs2 fragment was initially obtained from Jingxian DNA, digested with BstYI, self-ligated and amplified by inverse PCR using primers PMCi4 and PMCiD. Full-length Tcs2 was obtained by conventional PCR using primers PMC-G2 and PMC-S5. The sequence of the Ruby deletion allele (r) was obtained from OTA7 DNA using the same inverse PCR procedure and primers PMCi4 and PMCiD.

Expression Analysis of Ruby and Tcs1

Total RNA was extracted from 3 ml of juice of Moro, Tarocco, Navelina and Valencia fruit (all from CRA, Sicily) using a modified protocol described in Ancillo et al. (2007). DNAse-treated total RNA was further purified using the RNA Cleanup protocol (Qiagen) and retrotranscribed into cDNA using a High-Capacity cDNA Archive kit (Applied Biosystems). Quantitative real-time PCR was performed in optical 96-well plates with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). The PCR mixture (final volume 25 μl) contained 15 μl Power SYBR Green mix, 0.2 μM of each gene specific forward and reverse primers, 100 ng of cDNA sample, using the protocol for the Power SYBR Green PCR Master Mix (Applied Biosystems). The following standard thermal profile was used for all PCRs: 50° C. for 2 min; 95° C. for 10 min; 40 cycles of 95° C. for 15 s, and 60° C. for 1 min. Three replicates were assayed and a no-template negative control (H₂O control) was performed. The analyses used the relative quantification standard curve method.

Bioinformatic Analysis of LTR-Retrotransposons in Citrus

To estimate the proportion of the orange genome made up of full-length Tcs1-like elements the genome sequence of C. sinensis was scanned for Tcs1 sequences using the BLASTN program and the 5,413 bp Tcs1 transposon as the query sequence. Only hits with an e-value less than e⁻⁵⁰ and sequence identity to the Tcs1 sequence greater than 80% were used and filter masking was turned off.

The number of complete transposons identified was 78 where the BLAST high-scoring segment pair (HSP) covered greater than 70% of the length of the Tcs1 sequence. A further 47 LTR regions were identified that covered greater than 80% of the Tcs1 LTR region. Many of these hits to the LTR region had flanking hits to other regions of the transposon and were assumed to be full length transposons. Therefore, the estimated number of full length transposons is 125 and the proportion of the haploid genome sequence (296 mega bases; derived from the file called Csinensis_154.fa (July 2010) from the Phytozome website) occupied by these Tcs1-like elements is 0.23%.

To estimate the number of potentially active transposons, a Perl script was written to retrieve and translate the DNA sequence in the region of the open reading frame of the 78 complete transposons identified above. None of these translated regions were found to be without an internal stop codon therefore the corresponding elements are likely to be inactive.

Results

Identification of a Gene Encoding an R2R3 MYB Transcription Factor Expressed in the Fruit of Blood Orange Varieties

Anthocyanin biosynthesis is regulated mainly at the transcriptional level (Winkel-Shirley, 2001). A regulatory complex, composed of proteins of the Myb, Basic Helix-Loop-Helix (bHLH) and WD-Repeat (WDR) families of transcription factors, governs the expression of the structural genes required for anthocyanin biosynthesis, modification and transport (Ramsay and Glover, 2005; Butelli et al., 2008). In blood orange several anthocyanin biosynthetic genes show increased expression compared to blond orange (Bernardi et al., 2010; Coltrone et al., 2010; Licciardello et al., 2008). Variation in pigment intensity or tissue specificity is governed largely by the activity of the R2R3 Myb transcription factors in the complex (Espley et al., 2007; Geekiyanage et al., 2007; Walker et al., 2007; Takos et al., 2006; Schwinn et al., 2006; Kobayashi et al., 2004). Therefore, a partial cDNA fragment encoding the conserved Myb DNA binding domain, typical of the R2R3 Myb regulators of anthocyanin biosynthesis, was isolated from RNA from Moro flesh using degenerate PCR. This fragment was extended using 5′ and 3′ RACE PCR on cDNA prepared from the flesh of Moro fruit, to obtain a full-length cDNA and to map the start of transcription. We were unable to amplify an equivalent cDNA from fruit of common blond oranges and found no ESTs for the gene in the database collections. We named this R2R3 Myb gene, Ruby.

The Ruby cDNA encodes a 262 amino acid protein containing an R2R3 Myb domain with a signature motif for interaction with bHLH proteins from the clade 3f (DLX₂RX₃LX₆LX₃R, SEQ ID NO: 49; Lin-Wang et al., 2010; Zimmermann et al., 2004; Heim et al., 2003; FIG. 2A). It also has a conserved sequence motif KPXPR(S/T)F (SEQ ID NO: 50) in its C-terminal domain found in other R2R3 Myb regulators of anthocyanin biosynthesis (Lin-Wang et al., 2010; Stracke et al., 2001) Phylogenetic analysis revealed that Ruby clusters with members of R2R3 Myb subgroup 6, known to regulate anthocyanin biosynthesis in dicotyledonous plants (Lin-Wang et al., 2010; Bailey et al., 2008; FIG. 2B).

Ruby is a Regulator of Anthocyanin Biosynthesis

The ability of Ruby to activate anthocyanin biosynthesis was verified by its ectopic expression under the control of the constitutive CaMV 35S promoter in tobacco, where it resulted in visible purple-red pigmentation in undifferentiated callus and in developed tissues of regenerated plants (FIG. 3A). Co-expression of Ruby with the Delila and Mutabilis genes encoding bHLH transcription factors of clade 3f from Antirrhinum majus (Schwinn et al., 2006), known to interact with anthocyanin-specific Myb factors, enhanced pigmentation in tobacco (FIG. 3B). Ruby promoted stronger pigmentation in combination with Mutabilis than with Delila, suggesting some selectivity in the ability of the Ruby MYB protein to interact with different bHLH partners (FIG. 3B). These bioassays confirmed the functionality of Ruby as a regulator of anthocyanin biosynthesis. A cDNA encoding a clade 3f bHLH protein (CsMYC2) has been identified in orange (Coltrone et al., 2010). We showed, using transfection assays, that this bHLH protein will induce expression from the promoters of anthocyanin biosynthetic genes in combination with the Rosea1 R2R3 Myb regulator of anthocyanin biosynthesis from Antirrhinum majus (FIG. 9). These data support the idea that it is the expression of Ruby that limits anthocyanin biosynthesis in blood oranges because the CsMYC2 gene (encoding the bHLH partner in the MBW complex) is expressed at detectable levels in both blond and blood oranges (Coltone et al., 2010).

Expression of Ruby in Blood and Blond Orange Accessions

In blood oranges, Ruby expression was limited to the fruit (FIG. 4A). High levels of Ruby expression were detected in flesh and rind, including the white, spongy inner lining (albedo). Expression of Ruby was not detected in blond oranges (FIG. 4A). We tested the levels of Ruby expression in different accessions of blood orange and in hybrids between Moro and mandarin (OMO) and Tarocco and mandarin (OTA), produced in the experimental orchard of the Centro di Ricerca per l'Agrumicoltura e le Colture Mediterranee (CRA; Rapisada et al., 2009). The different orange accessions and the hybrids displayed a range of pigmentation levels when grown under equivalent conditions, from very high pigmentation (OTA9), medium pigmentation (OMO6, OTA23, OMO31, OMO15), low pigmentation (OMO28, OTA14, OTA20) through to no pigmentation (OTA41, OMO3, OMO5, OTA11, OTA17, OTA31, OTA35). The levels of Ruby transcript in the flesh of fruit grown under the same conditions at the field station were determined by qRT-PCR. There was a clear correlation between the levels of Ruby transcript and the levels of anthocyanin in fruit flesh (FIG. 4B). Expression of Ruby was also detected in ancient varieties belonging to the Sanguigno/Sanguinello group, generally characterized by light pigmentation of their flesh (FIG. 10).

Molecular Constitution of Ruby in Sweet Oranges

Blood orange is a derivative of sweet orange which is believed to be an interspecific hybrid between pummelo and mandarin (Li et al. 2011; Moore, 2001; Mabberley, 1997). To establish the genotypic constitution of different orange varieties at the Ruby locus, genomic DNA was extracted from leaves of pummelo, mandarin, and different sweet orange accessions. Pummelo contains two similar potentially functional alleles of Ruby, one of which showed complete sequence identity over 1.7 kb with the R allele identified in blond orange varieties. Mandarin was heterozygous at the Ruby locus. One allele contains a stop codon in the third exon of the gene which is predicted to result in an inactive Ruby protein. We termed this allele r-1. To characterise the second Ruby allele we used inverse PCR to identify a 2006 bp deletion which encompasses the first two exons of the Ruby gene and 1.4 kb of the region upstream (FIG. 6), creating a non-functional allele, which we named r-2. This second Ruby allele was present in mandarin and all orange varieties (FIG. 6). Our data from the Ruby locus confirm that sweet orange is a hybrid between pummelo and mandarin, and has inherited the R allele from pummelo and the r-2 allele from mandarin. Diploid mandarin carries two non-functional alleles of Ruby (r-1, r-2) implying that the generation of a ‘blood’ mandarin would be highly improbable. The haploid sequenced genome of mandarin (available on the World Wide Web at phytozome.net) carries the r-1 allele.

Molecular Differences at the Ruby Locus Between Blood and Blond Oranges

The Ruby gene was cloned from three blood (Sanguinelli, Maltaise Sanguine and Moro) and three blond (Navelina, Salustiana and Cadenera) varieties by PCR of genomic DNA. Sequence analysis revealed 100% nucleotide identity in the three exons and two introns that constitute the gene among the 6 varieties. The dramatic differences in expression of Ruby between blood and blond oranges suggested that they resulted from differences in the regulation of transcription of Ruby. The upstream regulatory regions of Ruby from Moro and Cadenera were isolated by chromosome walking and sequencing revealed an insertion of 501 nucleotides in Moro, 254 bp upstream of the initiating ATG in Ruby, compared with the otherwise identical Ruby promoter from the blond Cadenera orange (FIG. 5 and FIG. 6). The insertion showed sequence homology to the Long Terminal Repeats (LTRs) of the Copia family of retrotransposons, and included a 5 bp direct repeat, typical of LTR retroelement insertion sites (Kim et al., 1998).

DNA from the different blood orange accessions and from hybrids derived from the crosses between Moro (OMO hybrids) or Tarocco (OTA hybrids) and mandarin (Rapisada et al., 2009) was then mapped, amplified and sequenced (FIG. 5A). An accession of Moro from the UK National Citrus collection (Reeds Nursery Loddon, UK) had a larger insertion in the region upstream of the Ruby gene than the Moro accession from CRA, Acireale, Sicily. The same, larger insertion (termed R^D-1) was present in accessions of Tarocco from both CRA and the Reeds collection, in Maltaise Sanguine and Doppio Sanguigno representing older varieties of Italian blood oranges (Hodgson, 1967; Chapot, 1963) as well as in the Spanish Sanguinelli derived from Doblefina (FIG. 5A,B). The large insertions in Moro (Reeds), Tarocco, Maltaise Sanguine, Doppio Sanguigno and Sanguinelli were identical and corresponded to a complete retrotransposon, which we named Tcs1.

Tcs1 is 5413 nucleotides in length and shows all the features of a typical Copia-like long terminal repeat (LTR) retrotransposon. It contains an open reading frame, which encodes the proteins (Gag and Pol) required for the reverse transcription of the element and integration into the host genome, flanked by two identical LTRs of 496 nucleotides, identical to the solo LTR insertion in the Sicilian Moro (CRA). Because Tcs1 encodes complete Gag and Pol proteins it is likely an active retrotransposon and because the LTRs of Tcs1 at the Ruby locus are identical, it is likely that this is a very recent insertion.

A precise copy of Tcs1 is not present in the recently released sequence of the diploid C. sinensis genome (available on the World Wide Web at phytozome.net). Two closely related elements are present in the more accurately sequenced genome of a haploid C. reticulata (mandarin). They have identical LTRs to Tcs1 but differ in the non-coding region downstream of the 5′LTR. Several hundred Tcs1-like sequences can be identified in both species, the vast majority predicted to be inactive. In C. sinensis, LTR-retrotransposons have been estimated to constitute around 23% of the genome (Rico-Cabanas and Martinez-Izquierdo, 2007), but we calculate full length Tcs1-like copies, which have two LTRs available for recombination, constitute only about 0.23% of the genome and we could find no ‘active’ Tcs1-like copies with complete open reading frames in the available sequence.

Recombination Between LTRs of Tcs1 Gives Plants Chimeric at the Ruby Locus or Progeny with Just the LTR Insertion at the Ruby Locus

In addition to the insertion of the full Tcs1 element at the Ruby locus (which generated the dominant R^D-1 allele), the DNA from the leaves of Maltaise Sanguine and Sanguinelli accessions also contained versions of the Ruby locus with just the LTR inserted upstream, as shown by Southern blots and PCR (FIG. 5B). We deduced that these accessions are chimeric for insertions of the full element and the solo LTR. The intensity of the signal from the fragment containing the solo LTR from both Maltaise Sanguine and Sanguinelli suggests that these accessions are periclinal chimeras for the Ruby locus; a recombination could have occurred in the L1 or L2/L3 layers and then have been maintained over long periods of time because these varieties are propagated largely by grafting, as has been reported to occur commonly in other clonally propagated crops like grape (Pelsy, 2010).

Amongst the hybrids between Tarocco and mandarin (OTA hybrids) all those with pigmented fruit flesh had an insertion of the solo-LTR at the Ruby locus although a vegetative clone of the Tarocco parent plant used for the crosses had the full Tcs1 insertion (FIG. 5A). This showed that recombination can occur in the germline and suggested that it might be induced by meiosis. We also identified two accessions of Moro, one (Reeds UK) with the full Tcs1 retroelement insertion and the other, (CRA), with just the solo-LTR at Ruby indicating that unequal crossing over can also occur somatically (because blood orange is propagated by cuttings) and give rise to non-chimaeric progeny.

Retroelement Expression Controls the Expression of Ruby in Blood Orange

Retrotransposons can insert within or near transcriptionally active regions and can cause mutations by disrupting genes, altering their expression or driving genomic rearrangements (Shapiro, 2005; Feschotte et al., 2002; McClintock, 1984). We mapped the start of Ruby transcription by 5′ RACE PCR on cDNA prepared from RNA from Moro fruit flesh. The start of transcription mapped to an A, 551 nucleotides upstream of the initiating ATG of the Ruby gene. A TATA box was identified 32 bp upstream of the start of transcription within the LTR, whereas it was not possible to identify a TATA box in the sequence upstream of the Ruby gene in the R allele from blond oranges. The LTR also provided a 5′ donor splice site for the first intron in the Ruby transcript, the 3′ acceptor site being located within the sequences upstream of the Ruby open reading frame. A second intron detected in the Ruby transcript from Moro blood orange, was defined by donor and acceptor sites within the 5′UTR of the Ruby sequence (FIG. 7). We concluded that when Tcs1 is inserted at the Ruby locus, it must provide the regulatory sequences for initiating expression of the Ruby gene, since the transcription start site maps to the LTR.

Transcription of active retroelements, a prerequisite for transposition, is usually repressed by the host. However, activation in response to a variety of biotic and abiotic stresses is a common feature of most retrotransposons (Wessler, 1996). McClintock's theory of genome shock suggested that enhanced transposition under stress might represent an evolutionary strategy to increase the chances of survival under unfavorable conditions (McClintock, 1984). We therefore investigated whether the expression pattern of Ruby in blood oranges was a function of the expression of Tcs1 particularly in response to cold stress.

To assess the activity of Tcs1 and Tcs1-like elements, two sets of primers corresponding to the transcribed portion of the LTR or internal to the ‘Gag-Pol’ region of the element (FIG. 7) were used for qRT-PCR. The downstream primer for the transcript of the LTR corresponded to a sequence which is spliced from the Ruby transcript in the first intron in blood oranges (FIG. 7). Consequently, the Tcs1 LTR transcript levels we measured did not include those from the Ruby locus.

Tcs1 transcripts could be detected in fruit, but not in leaves, of blood oranges. Elevated levels of Tcs1 transcripts were observed in Tarocco and Moro blood varieties following storage of fruit in the cold, indicating that Tcs1 and Tcs1-like elements are activated by cold and ultimately are responsible for temperature-dependent anthocyanin accumulation in blood oranges (FIG. 7). The expression of Tcs1 matched well the change in transcript levels of Ruby in blood orange varieties in response to cold storage (FIG. 7). Since in Moro (CRA) the solo LTR controls expression of Ruby, cold-dependent transcriptional activation appears to be an intrinsic feature of Tcs1-like elements, irrespective of their association with the Ruby locus. Consequently, in the Sicilian blood orange varieties, the fruit-specific, cold-inducible production of anthocyanin (FIG. 7) is a feature of the regulation of the Tcs1 element, which controls expression of Ruby as a result of the positioning of the strong promoter provided by the 3′ LTR adjacent to the Ruby gene. Moro produces higher levels of anthocyanins than Tarocco. The higher anthocyanin production in Moro fruit is accompanied by higher expression of Tcs1. Consequently the selection of sports producing more deeply pigmented fruit flesh likely reflects selection for higher levels of Tcs1 transcription during the derivation of improved blood orange cultivars.

An Independent Blood Orange Accession from Jingxian, China

Today, blood oranges are grown in places as far apart as Japan, Australia, South Africa, Pakistan, California, China and Iran. However, the unreliable production associated with existing commercial blood orange varieties due to their cold dependency, means that the availability and consumption of blood orange on a global scale has declined in recent years (Zarba and Pulvirenti, 2005). There is considerable interest in identifying independent blood orange types that might be free from these production problems. We therefore looked amongst existing blood orange accessions for any independent events. Because of apomixis and a long juvenile phase, oranges, like most Citrus species, are almost exclusively propagated by grafting on selected rootstocks. Striking phenotypic diversity is due to the selection of superior or unusual branches derived from bud mutations. Our molecular analyses showed that Sanguinelli, which is a derivative of the Spanish Doblefina, shared a common origin with Sicilian blood oranges. Shamouti Blood Orange has been reported to be a chimera (Spiegel-Roy, 1979), possibly derived from grafting with Maltaise Sanguine (Hodgson, 1967), and is, therefore, also unlikely to represent an independent event.

Most of the blood orange cultivars grown in China are of direct or indirect Sicilian origin. However, one old variety, Jingxian, has been retained and is believed to be the only blood orange of Chinese origin (Yuan et al., 2008). Jingxian blood orange was first recorded in the Official Record for Huaihua Region in 1996. In 1965, when people began to select out this variety, the oldest tree they found in the countryside was about 70 years old. Consequently, the Jingxian blood orange has existed for at least 110 years.

Southern blot analysis indicated that Jingxian contains a DNA insertion in the Ruby locus of similar size to Tcs1 (FIG. 5A). However, inverse PCR showed this to be a different element, which we named Tcs2 (FIG. 6). Tcs2 is 5454 bp long, generates a 5 bp direct duplication upon insertion and is similar in sequence to Tcs1, except for the first half of the two LTRs that differ considerably. Like Tcs1, Tcs2 appears to be an active retroelement. In Jingxian blood orange, Tcs2 is inserted in the Ruby locus just 196 nucleotides upstream of the Tcs1 insertion site and 450 bp upstream of the Ruby ATG, but it is inserted in the opposite orientation to Tcs1 relative to the Ruby gene (FIG. 6).

The blood phenotypes in both Jingxian and the Sicilian group of blood oranges are therefore of independent origin showing that different members of the same family of retrotransposons may alter the expression of nearby genes through parallel but distinct mechanisms.

Despite smaller fruit size and higher seed content, confirming that Jingxian is a relatively distant cousin of Sicilian blood oranges, Jingxian fruit display the same pattern of cold-induced, fruit-specific anthocyanin accumulation as Sicilian and Spanish blood oranges, suggesting Tcs2 to be cold-inducible as well. For the LTR of Tcs2 to drive expression of Ruby, it must provide a bidirectional activator sequence. We mapped the start of transcription of Ruby in Jingxian juice to a position 321 nucleotides downstream of the Tcs2 insertion (FIG. 6). No TATA box was evident in the sequence ˜30 bp upstream of this transcriptional start site. Our data suggest that Tcs2 provides an upstream activating sequence that controls the expression of Ruby and the production of anthocyanin concordant with its own expression.

Activity of Copia-Like Retroelements in Citrus

A priori, the probability of independent gain-of-function mutations involving the same family of retroelements is low, especially given that Citrus varieties are almost exclusively vegetatively propagated. However, genome shock of the type caused by interspecific hybridisation does induce retroelement expression and transposition. It may be that the relatively recent origin (in terms of meiotic cycles) of sweet orange through interspecific hybridization between pummelo and mandarin (Li et al. 2011; Moore, 2001; Mabberley, 1997) induced accompanying high levels of retroelement activity which have been further selected during breeding of blood orange. Indeed, active retroelements may represent a major source of variation available to breeders of Citrus who depend on mutation-based differences that arise in buds (de Felice et al., 2008; Tao et al., 2005). Comparison of the DNA of the OTA hybrids and their parental lines showed new insertions of Tcs1-like elements following this interspecific cross (FIG. 8A). However, more striking was the high frequency of unequal crossing over between the LTRs of the full element to leave solo LTR insertions (FIG. 8B). This has also occurred at the Ruby locus in our accessions of Sanguinelli, Maltaise Sanguine, Moro and Tarocco. Sweet orange is a relatively recent derivative from an interspecific hybridization and the genome shock resulting from such hybridization may have stimulated higher levels of unequal crossing over. Interestingly, Parisod et al., (2009) highlighted significant structural changes occurring as a result of recombination involving retroelements, rather than transposition, during interspecific hybridization and allopolyploidisation in plants (Parisod et al., 2009).

DISCUSSION

The molecular analysis of the Ruby locus indicates that the genomes of pummelo (Citrus maxima) and mandarin (Citrus reticulata) combined to generate the genome of sweet orange (Citrus sinensis), confirming its reported hybrid origin (Li et al., 2010; Moore, 2001; Mabberley, 1997). At the Ruby locus the genetic contributions of the two parental species were equal and the functional allele of Ruby was provided by the pummelo parent. Although encoding a functional protein, this allele appears to be inactive in common blond orange, because we were unable to amplify any transcripts from this locus from any tissues of blond orange plants. Confirming its apparent lack of expression, no ESTs are available in the Citrus EST databases. Lack of Ruby expression may explain why anthocyanins, common pigments in most plant species, are rare in Citrus, and why mandarins, which carry only non-functional alleles of Ruby, never produce anthocyanins.

Our results indicate that all commercial blood orange varieties have a common origin. Anthocyanin pigmentation of fruit must have originated once either in a Mediterranean sweet orange or in a Chinese sweet orange which has since been lost. Citrus breeders have derived all the diversity in modern blood orange varieties from this original event. The molecular basis of the blood orange trait is retrotransposon-mediated transcriptional activation of the Ruby Myb gene. This is particularly clear in Sicilian blood oranges where the start of transcription of Ruby lies within the 3′ LTR of Tcs1. This provides a striking example of the role of transposable elements as controlling elements in the regulation of gene expression, adaptation to environmental stresses and genome evolution. Our discovery of a second independent insertion of a retroelement giving the same gain-of-function phenotype as in Sicilian blood oranges, illustrates the strength of the LTR as a promoter and also as an upstream activating sequence in the independent Jingxian blood orange. Both Tcs1 and Tcs2 insertions in Ruby give rise to induction of anthocyanin biosynthesis specifically in fruit, which is heavily influenced by environment. The cold dependency of anthocyanin production in blood orange results from the cold induction of retroelement transcription. The expression of Copia-like retrotransposons is determined by sequences within the LTRs which, in blood oranges, provide either a surrogate promoter with a TATA box and a transcriptional start as seen in commercial blood oranges, or an upstream activator sequence as seen in Jingxian orange. Consequently Ruby expression mirrors retroelement expression and is fruit-specific and cold-inducible.

Different accessions of blood orange demonstrate the high levels of recombination and transposition associated with the retroelements and suggest that they may be responsible for generating much of the diversity available to Citrus breeders (de Felice et al., 2009; Rico-Cabanas, and Martinez-Izquierdo, 2007; Tao et al., 2005). However, recombination between Tcs1 LTRs at the Ruby locus does not result in phenotypic changes in the levels of anthocyanins produced, confirming that the solo LTR carries all the information for the control of Ruby expression in Sicilian blood oranges. This is unlike the situation in grape. In grape, insertion of the Gypsy-like retrotransposon, Gret1, suppresses expression of a Myb gene (VvMYBA1) and it is believed that this insertion underpinned the development of white-skinned berries (Fournier-Level et al., 2010; Kobayashi et al., 2004). Recombination between Gret1 LTRs results in some restoration of Myb gene function and blush-skinned sports such as Chardonnay Rose and Flame Muscat (Pelsy, 2010).

The two independent blood orange derivatives, Jingxian and Sicilian blood oranges represent parallel gains of function, and therefore our results offer little hope of generating or identifying new varieties of blood orange that are free from the major limitation of cold dependency by conventional Citrus breeding methods. However, our improved understanding of the genetic and molecular basis of the blood orange trait could offer relatively straightforward solutions to the requirement for blood orange varieties with dependable production in warmer climates, through genetic engineering. Such strategies could provide new blood orange varieties suitable for the major areas of Citrus cultivation and could contribute significantly to increasing production of health-promoting blood oranges.

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The article “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one or more element.

Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1. A method for making a plant that is capable of producing a fruit with an increased level of anthocyanins, said method comprising modifying a plant so was to increase the expression of Ruby in the fruit of the plant, wherein a fruit produced by the plant comprises an increased level of anthocyanins when compared to a control fruit.

2. The method of claim 1, wherein modifying the plant comprises:

(a) introducing into at least one cell of the plant a polynucleotide construct comprising a promoter operably linked to a nucleotide sequence encoding Ruby, wherein the nucleotide sequence encoding Ruby comprises a nucleotide sequence selected from the group consisting of: (i) the coding sequence set forth in Sequence SEQ ID NO: 1, 3, 11, or 13, (ii) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, or 14, (iii) a nucleotide sequence comprising at least 75% identity to at least one of the full-length coding sequences set forth in SEQ ID NOS: 1, 3, 11, and 13, wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity, (iv) a nucleotide sequence encoding an amino acid sequence comprising at least 75% identity to at least one of the full-length amino acid sequences set forth in SEQ ID NOS: 2, 4, 12, and 14; wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity, (v) a fragment of any one of (i)-(iv), wherein the fragment encodes a polypeptide comprising Ruby transcription factor activity, and (vi) the nucleotide sequence of any one of (i)-(v), wherein the polypeptide further comprises at least one of the motifs, DLX2RX3LX6LX3R and KPXPR(S/T)F; or

(b) introducing into at least cell of the plant a polynucleotide construct comprising a promoter that is capable of driving the expression of an operably linked nucleotide sequence in the plant, whereby the polynucleotide construct integrates into the genome of the plant cell in operable linkage with a nucleotide sequence encoding a functional Ruby protein that is present in the genome of plant.

3. The method of claim 2, wherein the promoter preferentially drives expression in a fruit.

4. The method of claim 3, wherein the promoter comprises the nucleotide sequence set forth in SEQ ID NO: 15.

5. The method of claim 1, wherein the plant is a citrus plant.

6. The method of claim 5, wherein the promoter drives expression in the carpels of the fruit.

7. The method of claim 1, further comprising regenerating a plant comprising the polynucleotide construct.

8. The method of claim 7, further comprising growing the plant so as to produce at least one fruit.

9. The method of claim 8, wherein the fruit produced by the plant comprises an increased level of anthocyanins when compared to a control fruit.

10. The method of claim 1, wherein the polynucleotide construct is stably incorporated into the genome of the plant.

11. A transformed plant or plant cell comprising stably incorporated in its genome a polynucleotide construct, said polynucleotide construct comprising a promoter operably linked to a nucleotide sequence, wherein said nucleotide sequence comprises a member selected from the group consisting of:

(a) the coding sequence set forth in SEQ ID NO: 1, 3, 11, or 13;

(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, or 14;

(c) a nucleotide sequence comprising at least 75% identity to at least one of the full-length coding sequences set forth in SEQ ID NOS: 1, 3, 11, and 13, wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;

(d) a nucleotide sequence encoding an amino acid sequence comprising at least 75% identity to at least one of the full-length amino acid sequences set forth in SEQ ID NOS: 2, 4, 12, and 14; wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;

(e) a fragment of any one of (a)-(d), wherein the fragment encodes a polypeptide comprising Ruby transcription factor activity;

(f) the nucleotide sequence of any one of (a)-(e), wherein the polypeptide further comprises at least one of the motifs, DLX2RX3LX6LX3R and KPXPR(S/T)F; and

(g) a nucleotide sequence that is fully complementary to any one of (a)-(f).

12. The plant or plant cell of claim 11, wherein the plant is a citrus plant.

13. A fruit produced by the citrus plant of claim 12, wherein the fruit comprises the polynucleotide construct.

14. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:

(a) the coding sequence set forth in SEQ ID NO: 1, 3, 11, or 13;

(b) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 2, 4, 12, or 14;

(c) a nucleotide sequence comprising at least 75% identity to at least one of the full-length coding sequences set forth in SEQ ID NOS: 1, 3, 11, and 13, wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;

(d) a nucleotide sequence encoding an amino acid sequence comprising at least 75% identity to at least one of the full-length amino acid sequences set forth in SEQ ID NOS: 2, 4, 12, and 14; wherein the nucleotide sequence encodes a polypeptide comprising Ruby transcription factor activity;

(e) a fragment of any one of (a)-(d), wherein the fragment encodes a polypeptide comprising Ruby transcription factor activity;

(f) the nucleotide sequence of any one of (a)-(e), wherein the polypeptide further comprises at least one of the motifs, DLX2RX3LX6LX3R and KPXPR(S/T)F; and

(g) a nucleotide sequence that is fully complementary to any one of (a)-(f).

15. An expression cassette comprising a promoter operably linked to the nucleic acid molecule of claim 14.

16. A host cell comprising the expression cassette of claim 15.

17. The host cell of claim 16, wherein the host cell is a plant cell.

18. A plant comprising the expression cassette of claim 15.

19. A fruit, a fruit juice, or other food product comprising the expression cassette of claim 15.

20. A polypeptide encoded by the nucleic acid molecule of claim 14.