NUCLEOTIDE SEQUENCES ENCODING THE TOMATO LIGHT HYPERSENSITIVE PHENOTYPE, ENCODED PROTEINS AND USES THEREOF

Abstract: Nucleotide sequences of the tomato TDET1 (HP-2) gene are described, which sequences, if modified, result in a light hypersensitive phenotype. Vectors and uses for the production of transgenic plants are also described and transgenic plants so obtained.

Description: NUCLEOTIDE SEQUENCES ENCODING THE TOMATO LIGHT HYPERSENSITIVE PHENOTYPE, ENCODED PROTEINS AND USES THEREOF DESCRIPTION The present invention relates to nucleotide sequences encoding the tomato light hypersensitive phenotype, encoded proteins and uses thereof. In particular the present invention relates to nucleotide sequences encoding a protein, whose qualitative or quantitative modification and/or inhibition in plants induces high levels of carotenoids and/or flavonoids and/or chlorophylls, in comparison with wild-type plants; the invention also relates to the use of these nucleotide sequences for the production of engineered plants to be employed in the agro-industrial sector. Light is a critical environmental signal that controls many aspects of plant growth and development. It is perceived by a sophisticated series of photoreceptors : the phytochromes, which absorb red and far red light, the cryptochromes, which absorb blue and UV-A light wavelengths, and the UV-B receptors (Mustilli and Bowler, 1997) . Together with endogenous hormonal signals, these photoreceptors regulate the developmental changes known as photomorphogenesis. Photomorphogenesis is defined as the influence of light on plant development and comprises leaf and chloroplast development and the regulation of photosynthetic apparatus components, by means of the coordinated expression of both nuclear and cytoplasmic genes. Moreover, due to the light response, photoprotectant pigments such as flavonoids are also produced. The modifications occurring during photomorphogenesis have been characterized by studying light effects on Arabidopsis seedling development (von Arnim and Deng, 1996) . Light- grown Arabidopsis seedlings display short hypocotyls, open and expanded cotyledons and the expression of light- regulated genes which are responsible for flavonoid and chlorophyll biosynthesis (e.g. chalcone synthase, CHS; chlorophyll A/B binding protein, CAB) . Dark-grown seedlings display elongated hypocotyls, closed cotyledons and repression of light-regulated genes. In higher plants, the phytochrom.es are encoded by a gene family (e.g. PHYA-E in Arabidopsis, Sharrock and Quail, 1989; Clack et al . , 1994) and although they are the best characterized photoreceptors, relatively little is known about how the light signals perceived by phytochromes are transduced to the nucleus to activate the various developmental, physiological and molecular responses to light. Recently, biochemical studies using microinjection into cells of the phytochrome deficient a urea (au) tomato mutant, along with pharmacological studies in photomixotrophic soybean cell cultures, have implicated heterotrimeric G-proteins, cGMP, calcium and calmodulin as intermediates in phytochrome signal transduction pathways (Bowler and Chua, 1994; Mustilli and Bowler, 1997) . In parallel, several genetic screens have been developed to identify mutants potentially affected in light signal transduction (Chamowitz and Deng, 1996) . Most of the photo orphogenic mutants have been characterised in Arabidopsis and can be classified as either insensitive or constitutive mutants. Insensitive mutants display a light- blind elongated phenotype in the light. Some are mutated in the photoreceptors themselves, whilst others are presumed to encode positive regulators of light signal transduction pathways (Chamovitz and Deng, 1996; Chory et al., 1996; hitelam and Harberd, 1994) . Conversely, constitutive de- etiolated mutants (e.g. cop/det/fus/cpd) display light grown morphologies when grown in the dark together, in some cases, with the inappropriate expression of light regulated genes such as CAB and CHS (Millar et al . , 1994; Szekeres et al., 1996). The recessive nature of these mutations suggests that they are loss-of-function and that the wild- type genes are repressors of photomorphogenesis in darkness. However, although epistasis tests with phytochrome-deficient mutants have indicated that they function downstream of phytochrome, they are not specifically mutated in phytochrome signal transduction because many have altered tissue specificities as well as other additional phenotypes not directly related to light (Mayer et al., 1996; Chory and Peto, 1990; Millar et al., 1994; Szekeres et al., 1996). It is therefore not clear how COP/DET/FUS/CPD proteins function in the signal transduction pathways defined biochemically (Bowler and C ua, 1994) . A more targeted approach to identify specific components of signal transduction pathways specific for phytochrome could be the isolation of mutants with altered dynamics of light responses, rather than mutants with constitutive phenotypes in the absence of light. Several such light hypersensitve mutants have already been isolated in tomato (denoted hp-1 , hp-2, a tv, Ip; Kendrick et al . , 1994) . In particular, the recessive non-allelic hp-1 and hp-2 mutants have been characterized by their exaggerated light responsiveness, displaying higher anthocyanin levels (a flavonoid subgroup) , shorter hypocotyls and more deeply pigmented fruits than wild-type plants. These mutants were first identified in 1917 (Reynard, 1956) and in 1975 (Soressi, 1975) , respectively. Recently, hp-l w (Peters et al., 1989) and hp-2 J (Nan Tuinen et al., 1997) mutants have been isolated and identified as new hp-1 and hp-2 alleles, respectively. Because these phenotypes appear to be identical to those obtained by ectopic expression of phytochrome A { PHYA) in tomato (Boylan and Quail, 1989), it would appear that the hp mutation may affect fairly specifically phytochrome responses. The recessive nature of the mutations, coupled with results from epistasis tests of hp-1 with the phytochrome deficient tomato mutants aurea ( au) , phyA { fri ) , and phyB ( tri ) , have suggested that HP genes encode negative regulators of light signal transduction mechanisms, acting downstream of both PhyA and PhyB (Kerckhoffs et al., 1997). The fact that no counterparts of hp mutants have been isolated so far in Arabidopsis, along with the observation that in tomato anthocyanin production and the expression of photoregulated genes (e.g., CHS and CAB) is strictly light-dependent, indicates the importance of hp mutants for studying phytochrome-dependent signal transduction. Furthermore, microinjection-based studies using the au tomato mutant have shown that tomato is an excellent model system to map the role of individual components in the phytochrome activated signalling cascade (Bowler and Chua, 1994) . Therefore the identification and characterization of hp genes is likely to be very important for studying the regulation of photomorphogenesis in plants. The authors of the present invention have cloned the tomato HP-2 gene and have studied at the molecular level the role of the HP-2 protein during the modulation of photomorphogenesis and fruit development. The authors have found that the tomato HP-2 gene exhibits high sequence homology with the Arabidopsis DETl gene, which belongs to the above described constitutive COP/DET/FUS mutant group. Therefore the tomato HP-2 gene has been renamed TDETI . The authors have used Solanum lycopersicum (tomato) species plants, but those skilled in the art will recognize that the cloning could be repeated with no inventive efforts with other plant species, as but not limited to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae and ornamental species. The authors have cloned and sequenced the gene responsible for the tomato hp mutation {high pigment ) , which causes a light hypersensitive phenotype, thus enhancing carotenoid, and/or chlorophyll and/or flavonoid pigment levels. The gene is the first to be identified that causes such a mutant phenotype. hp mutants potentially have a direct application in the agro-industrial sector, for generating tomato fruits with high carotenoid and/or flavonoid contents. In particular, in tomato fruits of hp mutants, a high content of the carotenoid lycopene as well as other carotenoids and flavonoids has been observed (Thompson, 1955; Yen et al., 1997) . However, up to now the use of hp mutants in the agro-industrial sector, even if bred into various commercial varieties, has been impaired because of the fact that the hp mutation generates other undesirable phenotypes, such as reduced internode length and reduced plant vigour. The cloning of the TDETI gene and its use by means of gene transfer technologies offers considerable advantages with respect to conventional breeding techniques. It is possible to transfer genes suitable for agriculture between different species and to inactivate and/or engineer genes of the same species, even only in specific plant tissues. Furthermore gene transfer is a much more rapid technique than conventional breeding. The cloning of the TDETI gene now allows the production of engineered plants which exhibit all the favourable features of the hp mutation, with none of the undesirable side-ef ects. Such plants could belong to the Solanum genus, but those skilled in the art will recognize that, with no inventive effort, it is possible to transfer the gene, or parts thereof, subcloned in suitable expression vectors, into plants belonging to different genera, as e.g. but not limited to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae and ornamental species. It has been well recognized that a diet rich in lycopene and other carotenoids, or their administration in the form of pills, produce favourable effects on human health. As a matter of fact, carotenoids have antioxidant properties, β-carotene is a pro-vitamin A and lycopene is known to be an effective antitumoral agent (Bartley and Scolnik, 1995; Giles and Ireland, 1997; Hoffmann and Weisburger, 1997; Pappalardo et al., 1997; Pool-Zobel et al., 1997; Rock et al . , 1997; Sharoni et al., 1997; Stahl and Sies, 1996) . Therefore the engineered plants of the present invention can be advantageously utilized as a source of such compounds, either as fresh or processed foods or as nutraceuticals . Furthermore hp-2 mutants have high levels of flavonoids such as anthocyanins (Von Wettstein-Knowles, 1968), which are also considered to be excellent antioxidants and which exhibit in some cases antitumoral properties (Fotsis et al . , 1997; Rice-Evans et al., 1997). Furthermore some flavonoids exhibit a role in plant protection against pathogenic agents and UV light irradiation (Shirley, 1996) . Therefore the manipulation of TDETI activity can be used to modify carotenoid and/or flavonoid content in several plant species (e.g., tomato, pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, and pomaceae) for biotechnological uses in both the biomedical and agro-industrial sectors. Furthermore the manipulation of TDETI gene expression can be used to modify the anthocyanin and carotenoid content in ornamental species for the achievement of new colour variants (Griesbach, 1984). In addition, because it has been recognized that a high content of carotenoids improves resistance to Norflurazon-type herbicides, a further application of the TDETI gene is the production of transgenic plants by selection using herbicides rather than antibiotic compounds (Misawa et al., 1993). Furthermore in the same plant it is possible to combine a modified TDETI activity with mutations such as rin, nor and Nr, which interrupt the fruit ripening process, or with biosynthetic genes of the carotenoid biosynthesis pathway (Bartley and Scolnik, 1995) , to obtain varieties exhibiting new qualitative characteristics for agro-industry. The attainment of an hp mutant phenotype by means of a biotechnological approach can be carried out advantageously by means of the inhibition of TDETI activity. Currently the best method for reducing gene expression is through the introduction, by gene transfer, of the antisense sequence of the same gene or part thereof under the control of appropriate regulatory sequences. The use of such techniques in plants has been carried out in several existing examples (Oeller et al., 1991; Penarrubia et al., 1992), but those skilled in the art will recognize that alternative techniques can be used, without departing from the scope of the present invention. Furthermore, by using specific vectors such as, for example, but not limited to, pE8mGFP4, which contains regulatory sequences of the tomato ES gene (Fig. 9) (Deikman and Fischer, 1988), the inactivation of the TDETI gene can be specifically modulated in the tomato fruit. Those skilled in the art will recognize that other regulatory sequences, e.g., originating from the polygalacturonase gene { PG) (Nicholass et al . , 1995), can be used in the place of the E8 gene promoter to obtain specific tissue modulation of TDETI gene expression. Within the scope of the present invention the term "light hypersensitive phenotype" means reduced plant growth associated with high levels of carotenoids and/or chlorophylls and/or flavonoids. The term "protein or functional parts thereof, responsible for the light hypersensitive mutant phenotype" means an amino acid sequence which, if structurally or otherwise altered, induces a light hypersensitive phenotype in a plant. Therefore one object of the present invention is a nucleic acid comprising a nucleotide sequence encoding a protein, or functional parts thereof, which, if modified, is responsible for the light hypersensitive mutant phenotype in Solanum lycopersicum plants, said phenotype including reduced plant growth associated with high levels of carotenoids and/or chlorophylls and/or flavonoids. Nucleic acids encoding proteins which are homologous to proteins of the Arabidopsis COP/DET/FUS family, which when modified result in a light hypersensitive phenotype, are within the scope of the present invention. Preferably the nucleic acid comprises the nucleotide sequence encoding the TDETI protein, or functional parts thereof. More preferably the nucleic acid comprises a nucleotide sequence encoding the protein having the amino acid sequence of SEQ ID No. 2 or functional portions thereof. More preferably the nucleic acid has a nucleotide sequence comprised in SEQ ID No. 1, more preferably from nt. 149 to nt. 1720. Alternatively the nucleic acid has a nucleotide sequence complementary to SEQ ID No. 1, or parts thereof. In one aspect of the invention the nucleic acid of SEQ ID No. 1 comprises a mutation which is able to induce the light hypersensitive phenotype; preferably at least a C→T substitution in position 1640; alternatively the nucleic acid of SEQ ID No. 1 is deleted at least from nt . 1581 to nt. 1589. A further object of the present invention is an expression vector including, under the control of an active and inducible plant promoter, the nucleic acid of the invention. Preferably the promoter is active only in some plant organs, more preferably in fruits. Preferred vectors are able to drive the transcription of an antisense RNA, for example pBIN-E8-HP2-ASl and pBIN-E8-HP2-AS2. A further object of the invention is the use of the vectors of the invention for producing transgenic plants which comprise the nucleic acid, under the control of specific regulating sequences, preferably in preselected plant organs. Plants can belong to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae and ornamental species. A further object of the invention is a transgenic plant, or parts thereof, achievable by transformation with the vectors of the invention. Plants can belong to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae and ornamental species. Plant genetic transformation techniques are known to those skilled in the art and comprise, but are not limited to transformation by Agrobacterium, electroporation, microinjection, or bombardment with DNA coated particles (Christou, 1996) . In a further aspect the invention includes a protein, or functional parts thereof, whose modification is responsible for the light hypersenstive phenotype in Solanum lycopersi cum plants. Proteins homologous to the Arabidopsis COP/DET/FUS family are within the scope of the present invention, provided that, when modified, they result in a light hypersensitive phenotype. Preferably the protein comprises the amino acid sequence of SEQ ID No. 2 or parts thereof. In one aspect of the invention the protein comprises a modification which is able to induce the light hypersensitive phenotype; preferably at least a modification in its C-terminal portion; more preferably a replacement of proline at position 498, most preferably serine as a substitute for proline; alternatively a deletion of at least one amino acid, preferably at least of three amino acids, more preferably from aa. 478 to aa. 480 of SEQ ID No. 2. The present invention will be described by reference to explanatory, but not limiting, examples, wherein reference will be made to the following figures: Figure 1. Phenotypes of wild type, hp-2, and hp-2 J tomato seedlings grown in the presence or in the absence of light, a) Hypocotyl length of seedlings grown in the absence (Dark) and in the presence of light (Light) . b) Content of anthocyanins in the hypocotyls (dashed bars) and in the cotyledons (empty bars) of seedlings grown in the light. Data represent average values from ten seedlings. Following a three day pre-germination period in darkness, seedlings were grown either in darkness (Dark) or in a 16 hour light, 8 hour dark photoperiod (Light) for 6 days. Figure 2. TDETI {HP-2) cDNA nucleotide sequence (SEQ ID NO. 1) . The sequence encoding the TDETI protein is depicted in capital letters. The sequence of the RFLP CT151 marker covers nt. 830 to nt . 2000. Figure 3. Amino acid sequence of the TDETI (HP-2) protein (SEQ ID No. 2) . Amino acids are depicted in the standard one letter code for amino acids. Figure 4. Exon-intron structure of the tomato TDETI {HP-2) gene and sites of mutations in hp-2 and hp-2 mutant alleles. Stippled boxes indicate exons . Exons correspond to the following sequences of SEQ ID No. 1 (Figure 2) : Exon 1: nt. 149 to nt . 220; Exon 2: nt . 221 to nt . 648; Exon 3: nt . 649 to nt. 877; Exon 4: nt . 877 to nt . 1012; Exon 5: nt . 1013 to nt. 1081; Exon 6: nt . 1082 to nt . 1138; Exon 7: nt . 1139 to nt. 1219; Exon 8: nt . 1220 to nt . 1384; Exon 9: nt . 1385 to nt. 1480; Exon 10: nt . 1481 to nt . 1580; Exon 11: nt. 1581 to nt . 1720; b) Donor and acceptor splice sites of intron 10 from the wild type and the hp-2 mutant. Brackets indicate splice sites, dots indicate internal intron sequence (not shown) , and amino acids are shown in the one letter code. The asterisk in the hp-2 sequence denotes the site of the mutation. Figure 5. Alignment of the amino acid sequences of S. lycopersicum TDETI, Arabidopsis DETl, and deduced mammalian EST amino acid sequences with homology to DETl (determined using Clustal method; Higgins and Sharp, 1988) . The putative bipartite NLS is overlined. The amino acids missing in the hp-2 mutant are indicated with asterisks, and the amino acid substitution in hp-2J is denoted by a plus sign. The mammalian sequences are a compilation of derived amino acid sequences from mouse and human ESTs (GenBank accession numbers AA756238, AA236057, AA050184, and W64359) . Boxed residues indicate conserved amino acids, dashes indicate arbitrary insertions. Figure 6. Effect of cytokinins on wild type tomato seedlings . (a) and (b) Hypocotyl length (cm) and anthocyanin content (per mg fresh weight [FW] ) , respectively, of wild type tomato seedlings grown in the presence of 0, 1, 5, 20, 100, and 500 μg/L benzoaminopurine . Seedlings were grown at 25 °C for 5 days in absolute darkness (Dark) or in a 16-hr light 8-hr dark photoperiod (Light) . In (a) values are the mean of 10 seedlings. Highly similar results were obtained with the cytokinin zeatin (data not shown) . Figure 7. Phenotype of the au hp-2 double mutant phenotype. Hypocotyl length (cm) and anthocyanin accumulation (per seedling) in wild type, au, hp-2, and the au hp-2 double mutant are shown. Seedlings were grown at 25 °C for 6 days in a 16-hr light 8-hr dark photoperiod. Values are the mean of 15 seedlings, and the experiments were repeated three times. Figure 8. Gene expression in wild type, hp-2. and hp- 2 seedlings. Seedlings were grown at 25 °C for 5 days in absolute darkness (D) , followed by 2 days in continuous white light (L) . The RNAs were extracted from whole seedlings (dark) or from cotyledons (C) and hypocotyls (H) (light) . Modifications in gene expression in hp-2 and hp-2J seedlings compared with wild type seedlings (wt) are shown to be principally light-dependent. Ten microgram samples of total RNA were loaded on gels and analyzed for expression of CHSl { CHS) , CAB6 { CAB) , PRl -lbl { PR) genes following RNA gel blotting. 30S rRNA is shown as a control for loading. Figure 9. Binary plasmids to be transferred to plants . Materials and Methods Plant material and growth conditions The hp-2 and hp-23 exaggerated photoresponse mutants, the au hp-2 double mutant, and the corresponding wild type tomato seeds { Solanum lycopersi cum cv Money Maker) were kindly provided by R. E. Kendrick and M. Koornneef (Wageningen Agricultural University, the Netherlands) . Seeds were surface sterilized and directly sown in magenta boxes (Sigma) containing 4.3 g/L Murashige-Skoog salts (Sigma) and 0.8 % agar. After 2 days pregermination in darkness, seedlings were grown at 25° C either in a 16 hour light, 8 hour dark photoperiod or in continuous dark, as appropriate. For cytokinin treatment, seeds were sown in the presence of different concentrations of benzylaminopurine (Sigma) . Anthocyanin assay Anthocyanins were extracted from cotyledons, hypocotyls, and whole seedlings with 0.5 mL acidified (1% HCl) methanol for 48 hr in darkness with shaking. The extracts were separated by the addition of 0.4 mL of H 2 O and 1 L of chloroform, followed by centrifugation for 5 min at 3,000 rpm. The absorbance of the upper phase was determined spectrophotometrically at 535 nm (A 535 ) , and the anthocyanin content was calculated as (As 35 )/mg fresh weight or (A 535 ) /seedling. Carotenoid and Chlorophyll assay In order to determine the content of carotenoids and chlorophyll in the tomato fruit, pericarp sections of unripe (25 days after pollination) or ripe (50 days after pollination) fruit were weighed and incubated for 48 hours at 65°C in DMSO (dimethyl sulfoxide) in the absence of light. The contents of carotenoids and chlorophylls were determined by HPLC and values are reported as μg/g fresh weight. DNA and RNA Extraction DNA and RNA extractions from leaf samples were carried out according to Dellaporta et al . (1983) and Verwoerd et al . (1989), respectively. RNA gels (10 μg per lane) were blotted onto Hybond N+ membranes (Amersham) and hybridized with random primed probes (see below) . Hybridization was performed for 24 hr at 50°C in phosphate buffer (7% SDS, 0.5M Na 2 HP0 4 , pH 7.2, ImM EDTA) , followed by 20-min washes in 40mM NaP0 4 pH 7.2, 1% SDS, and ImM EDTA. Probes were the tomato chalcone synthase gene CHSl (O'Neill et al., 1990), chlorophyll a/b binding protein gene CAB6 (Piechulla et al . , 1991) and pathogenesis-related protein gene PR-lbl (Tornero et al., 1997) . All RNA gel blots were repeated at least twice and using different samples. Isolation and sequence of genomic clones, cDNAs and PCR products 5' amplification of cDNA was performed using the RACE system (Gibco/BRL) on total tomato leaf RNA with the oligonucleotide 5' -CATCAACACTGCCAAAC-3' (SEQ ID No. 3), derived from the CT151 RFLP marker sequence. A 0.8-kb Polymerase Chain Reaction (PCR) product, containing the 5' end of the TDETI cDNA, was obtained after two different amplification reactions with Taql polymerase (Perkin Elmer) using two CT151-derived nested primers: 5'- GAAAGCAGCCGTTGCT-3' (SEQ ID No. 4) and 5'- AGTTCATCATCTTCACGGC-3' (SEQ ID No. 5), with the provided anchor primer (Gibco/BRL) The 0.8kb PCR fragment was directly sequenced on both strands with ther osequenase (Amersham) . Total cDNA from wild type tomato (cv. Money Maker) and the corresponding hp-2 and hp-2 mutants were obtained by reverse transcription using avian myeloblastosis virus reverse transcriptase (Promega) of poly (A) mRNA isolated from leaves using oligo(dT) Dynabeads (Dynal) . From these samples the TDETI {HP-2) cDNA sequence was PCR amplified with the specific oligonucleotides : 5'-GTATGATTCACTAGTTTAATGCTGCTGAAAG-3' (SEQ ID No. 6) and 5'-CCCATACTAGTCGTCTTGGCACTCTATCAAG-3' (SEQ ID No. 7), using the Expand High Fidelity system (Boehringer Mannheim) . Subsequently the amplification product was subcloned in pBluescript as a Spel fragment and 4 independent clones were sequenced on both strands. Tomato genomic libraries in λ-DASH and λ-FIXII (kindly provided, respectively, by Jim Giovannoni, Texas A&M University, College Station, TX, and the Tomato Genome Center, Rehovot, Israel) were screened using standard methods (Sambrook et al . , 1989) with a 32 P-labelled CT151 fragment as a probe. Overlapping fragments from different recombinant phages were subcloned in pBluescript SK+ for sequencing of both strands. The 5'-GAAGGTAATTTTATATTAAACATAGAATAGA-3' (SEQ ID No. 8) and 5' -GTGATTTCTAGGTTGATTTCAATCTAGA-3' (SEQ ID. No. 9) oligonucleotides were used to amplify the HP-2 gene 3' sequence from genomic DNA of the hp-2 mutant. The 1,300 bp amplification product was directly sequenced with the primer 5' -CAAATCGGTAACATAT-3' (SEQ ID No. 10) using a Thermosequenase kit (Amersham) . Binary plasmids for plant transformation DNA fragments containing the tomato fruit-specific E8 promoter (Deikman and Fischer, 1988) were PCR amplified from S . lycopersicum total DNA with the following nucleotides: 5' -GGGGAAGCTTTTTCACGAAATCGGCCCTTA-3' (SEQ ID No. 11) and 5' -CCCGGATCCTTCTTTTGCACTGTGAATGATTAG-3' (SEQ ID No. 12). The 1.2kb amplification products were subcloned as Hindlll-BamHI fragments in the binary expression vector pmGFP4, derived from pBI121 (Bevan, 1984; Jefferson et al . , 1987; Haseloff et al . , 1997) in place of the 35S promoter, obtaining the plasmid pE8mGFP4. The mGFP4 gene was then excised and the inverted or complementary sequence of the HP-2 gene, or parts thereof, was inserted, as BamHI-SacI fragments, obtaining the plasmid pBIN-E8-HP2-ASl and pBIN- E8-HP2-AS2, respectively (Figure 9) . Results hp-2 mutant phenotypes hp-2 mutant seedlings exhibited an exaggerated light response with respect to wild type seedlings. Particularly, hypocotyl length was reduced (Figure la) and the content of anthocyanin pigments in the cotyledons was higher than in the wild type (Figure lb) . Such phenotypes were strictly dependent on the presence of light; in fact in the absence of light the length of hypocotyl was not considerably different to the wild type (Figure la) and anthocyanins were not produced (data not shown) . The hp-2 3 mutant exhibited a stronger phenotype than the hp-2 mutant, with reference both to hypocotyl length and the content of anthocyanins (Figure 1). Fruits obtained from plants of hp-2 and hp-2 mutants also exhibited exaggerated light responses. In the unripe fruits from hp-2 and hp-2 mutants the content of chlorophyll was about five times higher than in fruits from wild-type plants, whereas in mature fruits the total content of carotenoids was about twice that of fruits from wild-type plants (Table 1) . Table 1 Chlorophyll and carotenoid content of tomato fruits from wild type, hp-2 and hp-2 mutants. Genotype Total Chlorophylls Total Carotenoids (ripe fruit) (unripe fruit) g/g fresh weight ug/g fresh weight wild type 20 60 (Money Maker) hp-2 90 124 hp-2 3 115 137 The Phenotype of the Tomato hp-2 Mutant is Caused by Mutation in DETl The hp-2 mutation has been mapped previously using restriction fragment length polymorphism (RFLP) analyses of a segregating population derived from a cross between the hp-2 mutant { S . lycopersi cum) and S. pennellii . Mapping data with a second backcross (BC-2) population indicated a position close to the centromere of chromosome 1, within a cluster of several RFLP markers (Balint-Kurti et al . , 1995; Van Tuinen et al . , 1997; Broun and Tansksley, 1996). The authors of the present invention have found that the CT151 RFLP marker (Tanksley et al . , 1992), included in this cluster, had high homology with the Arabidopsi s DETl gene, mutations in which were previously identified as causing the deetiolated phenotype of detl mutants. Comparison of CT151 with Arabidopsis DETl revealed that CT151 has homology to the 3' end of DETl . The authors of the present invention isolated a full-length cDNA encoding TDETI using 5' rapid amplification of cDNA ends (Loh et al., 1989). Subsequently, the TDETI gene was isolated from genomic libraries in λ-DASH and λ-FIXII. Alignment of the TDETI cDNA and genomic sequences revealed the presence of 10 introns, which is similar to the number found in Arabidopsis (Figure 4a) . Nine introns are located in the same positions as those of the Arabidopsis DETl gene, whereas intron 2 of TDETI is not present in the Arabidopsis ho olog (Figure 4a) (Pepper et al . , 1994). Comparative protein sequence analysis between Arabidopsis DETl and TDETI show 81.3% similarity and 74.8% identity (Figure 5) . No major differences are present between the sequences, except for a small deletion of 16 amino acids at the centre of TDETI , suggesting that the tomato and Arabidopsis genes are true homologs. A DETl homolog is not present in the yeast genome or in any prokaryotic genome sequenced to date (data not shown) . However, mouse and human expressed sequence tags (ESTs) with homology to DETl have been identified (Figure 5) , as well as a Drosophila homolog. Considering the presence of a nuclear localisation signal (NLS) (Robbins et al . , 1991) in the Arabidopsis DETl amino acid sequence, and observing that the DET1-GUS chimeric protein is localised in the nucleus (Pepper et al., 1994), DETl in Arabidopsis may be responsible for the transcriptional repression of photomorphogenesis in the absence of light. Considering the homology with HP-2 it is possible for the latter to have an analogous function in tomato. The TDETI protein has been found to be localized in the nucleus (data not shown) . TDETI -encoding cDNAs from the wild type, and hp-2 and hp-2l mutants were amplified with specific primers from leaf mRNA. Amplified fragments were subcloned in pBluescript and 4 independent clones were sequenced on both strands. In hp-2 the authors identified a substitution involving T in the place of C in exon 11 (nt. 1640) that causes a replacement of proline by serine in the protein C- terminal region (aa. 498) (Figure 4a) . In the hp-2 mutant the authors identified an alternative splicing site for intron 10, which causes a three amino acid deletion (Gly, Pro, Glu) at the start of exon 11, in the second NLS domain of the protein (aa. 478-480) (Figure 4a) . To find the mutation site which was responsible for the alternative splicing, the authors sequenced the 3' end of the TDETI gene from the hp-2 mutant, using a fragment obtained by PCR amplification of genomic DNA. A replacement of AG by TG in the consensus 3' splice junction of intron 10 was identified (Figure 4b) . These results demonstrate that the "high pigment" phenotype of the hp-2 and hp-2 J tomato mutants is caused by mutation of the TDETI gene mutation. Sequencing of the TDETI cDNAs derived from the hp-2 mutant showed that the mutation produces two different splicing products from intron 10 (Figure 4b) , suggesting the presence of a limited amount of wild-type TDETI protein and that the hp-2 mutant is not an inactive allele. This is consistent with the observation that the hp-2 mutant allele is weaker than the hp-2 j allele (Figure 1 and Table 1) . Whether the hp-2 : mutation is null awaits further analysis, although the mutated proline residue in the hp-2 j mutant is conserved in both the plant sequences. However, because the hp-2 mutation is situated in the C-terminal domain of TDETI, as is the mutation in the Arabidopsis detl -5 mutant allele, which has a clearly de-etiolated phenotype (Pepper et al., 1994), it is apparent that the hp-2 mutant phenotypes are not comparable with the detl mutant phenotype in Arabidopsi s . Furthermore, clearly weak alleles of det-1 in Arabidopsis, eg. detl -1 , display visible dark phenotypes . Comparison of Tomato hp-2 and Arabidopsis detl Mutants As is clear from the results presented, hp-2 mutants are phenotypically different from detl mutants. Most conspicuously they do not display dark phenotypes, such as reduced hypocotyl length, opened apical hooks, or enlarged cotyledons, whereas these were selection criteria for the isolation of det and cop in Arabidopsis (Chory et al . , 1989; Deng et al . , 1991). Conversely, detl mutants can even develop true leaves and floral buds in prolonged darkness. No such phenotypes can be observed in hp tomato seedlings grown in the dark. Arabidopsis detl mutants are also characterized by high level expression of light-regulated genes such as CHS and CAB in the dark, whereas in the light, CHS and CAB gene expression are similar to that observed in wild-type plants (Chory et al . , 1989). To examine the effects of hp-2 and hp-2J mutations on gene expression, the authors have performed RNA gel blot analysis of light-regulated gene expression using CHS and CAB gene fragments as probes. In agreement with the weak phenotypes of hp-2 and hp-23 mutant seedlings grown in darkness, no dramatic alteration of CHS and CAB gene expression was observed. Nonetheless, CHS and CAB mRNA levels were slightly higher in the hp-2J allele when compared with hp-2 and wild-type seedlings (Figure 8) . Interestingly, CHS mRNA appeared to be of a slightly smaller size than that found in light-grown material, perhaps indicating a differential light-dependent splicing. Consistent with the exaggerated photoresponsiveness of hp-2 and hp-2J mutants, CHS mRNA levels were significantly enhanced compared with wild-type seedlings following light irradiation for 48 hr (Figure 8). hp-2 seedlings contained higher expression levels of CHS than hp-2, and CHS mRNA was particularly abundant in hypocotyls. In contrast to CHS, CAB mRNA levels were higher in cotyledons than in hypocotyls, and were slightly lower in light-exposed hp-2 and hp-2J mutant seedlings when compared to the wild type (Figure 8) . In summary therefore, in tomato hp-2 mutants dark expression of CHS and CAB genes is only very slight and deregulation of gene expression is essentially light- dependent. This is in strong contrast to the CHS and CAB gene deregulation characteristics of Arabidopsi s detl mutants (Pepper et al., 1994). detl seedlings have been also reported to display a strongly enhanced expression of stress-related genes such as those encoding pathogenesis-related (PR) proteins and glutathione reductase (Mayer et al., 1996) To examine whether this was also the case in hp~2 and hp-23 mutants, RNA gel blots were hybridized with a probe encoding tomato PR-lbl (Tornero et al., 1997). Although hypocotyls of hp-2 seedlings reproducibly displayed PR-lbl gene expression at slightly higher levels compared to wild-type seedlings, this effect was very weak compared to that observed in Arabidopsi s detl mutants and has never been observed in hp- 23 mutants (Figure 8) . Despite these above-mentioned differences, plastid development in darkness in the cotyledons of hp-2 and hp-2J seedlings was found by the authors to be similar to that observed in Arabidopsis detl mutants (data not shown; Chory et al. , 1989) . Cytokinin can Phenocopy the hp Mutant Previous observations in Arabidopsis have shown that a detl phenotype can be phenocopied by the exogenous application of cytokinin (Chory et al . , 1994). To determine the effects of cytokinin treatment in tomato, the authors treated wild-type seedlings with different concentrations of cytokinin in the dark and in the light (Figure 6) . Cytokinin was found not to phenocopy a detl mutation in tomato. In dark-grown seedlings, although hypocotyls are shorter in the presence of cytokinin, apical hook opening, cotyledon expansion, and anthocyanin biosynthesis were not observed (Figure 6) , even after prolonged periods (up to three weeks) (data not shown) . However, in the light, cytokinin can phenocopy the hp mutation: seedlings displayed shorter and thicker hypocotyls and accumulated high levels of anthocyanin (Figure 6) . These results therefore indicate that at least as far as the effects of cytokinin are concerned, the hp mutation in tomato is equivalent to the detl mutation in Arabidopsis . Such a supposition is consistent with the fact that no constitutive de-etiolated mutants have heretofore been identified in tomato and CHS gene expression and consequent biosynthetic build-up of anthocyanin pigments is strictly dependent on the light, whereas in Arabidopsi s anthocyanins can be produced also in the absence of light (Mustilli and Bowler, 1997) . Double Mutant Analyses Double mutant analysis with detl and phytochrome- deficient mutants in Arabidopsis indicate that the detl mutation is completely epistatic to photoreceptor mutations (Chory, 1992) . This has been interpreted as meaning that DETl acts downstream of phytochrome. To determine the relationship between TDETI and phytochrome function, we examined the effects of the hp-2 mutation in the aurea mutant background, a phytochrome chromophore-deficient mutant (Terry and Kendrick, 1996) . In contrast to its counterpart double mutant in Arabidopsis, au hp-2 is similar to the single mutant au . For example, hypocotyls are elongated and anthocyanin accumulation is limited (Figure 7) . Although in genetic terms this result therefore infers that au is epistatic to hp-2, the authors nonetheless consider it more likely that TDETI acts downstream of phytochrome, as proposed for Arabidopsis, but that its activity as a negative regulator is strictly dependent upon the presence of active phytochrome. This requirement is clearly not observed in Arabidopsis (Chory, 1992) . The small but significant reduction in hypocotyl length and the small increase in anthocyanin found in the au hp-2 double mutant compared with au (Figure 7) is likely therefore to be a result of hypersensitivity caused by the hp-2 mutation towards the low amounts of functional phytochrome present in the au mutant, which is estimated to be about 3% wild-type levels (Adams et al . , 1989; Parks et al, 1987) . Therefore the hp-2 phenotype in tomato is strictly dependent on the presence of phytochrome, whereas in Arabidopsis the detl mutation is independent of the presence or absence of functional photoreceptors. The absence of de-etiolated mutants in tomato and the strict light- and phytochrome- dependence of the hp mutant phenotype may suggest that in tomato TDETI function is either redundant or that the signal transduction pathways regulating its activity operate in different ways in the two plants (Mustilli and Bowler, 1997) . Because by Southern analysis the authors have shown that in tomato, like in Arabidopsis (Pepper et al . , 1994), only one DETl homologous gene is present (data not shown) , the latter hypothesis seems to be more likely. Based upon the author's findings, it is likely that only a few key regulators will be found to be responsible for controlling light responses in all higher plants. Therefore it would be very interesting to clone other tomato genes encoding proteins homologous to COP/DET/FUS and examine whether modification of their activity also results in light hypersensitive phenotypes. Binary plasmids for modulation of TDETI activity In order to generate tomato plants with an hp-2 phenotype specifically in the fruit, the authors constructed plasmids which are able to produce the TDETI antisense RNA sequence, or portions thereof, only in the fruit by using the regulatory sequences of the tomato E8 gene (Figure 9) . Bibliography - Ada se, P., et al. (1989) Photochem. 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SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Stazione Zoologica "Anton Dohrn" (B) STREET: Villa Comunale (C) CITY: Naples (E) COUNTRY: Italy (F) POSTAL CODE (ZIP) : 80121 (ii) TITLE OF INVENTION: Nucleotide sequences encoding the tomato light hypersensitive phenotype, encoded proteins and uses thereof (iii) NUMBER OF SEQUENCES: 12 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2000 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 149..1720 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTTCCCTCTT AGACTTTATC GATCCTAATT CGAGCCCTCC TTTTTTCAAT CAATTATCAA 60 TTTAGTCCTA CTGCGATTTT GATATGTATG ATTCACAATT TTAATGCTGC TGAAAGCAAT 120 TATATAAAAG CTGAAACATT TTGCACTG ATG TTC AAA ACT AAC AAT GTT ACC 172 Met Phe Lys Thr Asn Asn Val Thr 1 5 GCC AGG CTT TTT GAG CGC CAG ATT TGC ACC CCT GCT CCT GGC ACC AGC 220 Ala Arg Leu Phe Glu Arg Gin lie Cys Thr Pro Ala Pro Gly Thr Ser 10 15 20 ATC CAT CGT GCC AGA AGA TTT TAT GAG AAC GTT GTA CCA AGT TAT ACC 268 lie His Arg Ala Arg Arg Phe Tyr Glu Asn Val Val Pro Ser Tyr Thr 25 30 35 40 ATA TAC GAT GTT GAA TGT CCC GAC CAT TCA TTT CGC AAG TTC ACG GAT 316 lie Tyr Asp Val Glu Cys Pro Asp His Ser Phe Arg Lys Phe Thr Asp 45 50 55 GAC GGT CTA TAT TTT GTA AGT TTC AGC CGA AAC CAT CAG GAT CTG GTT 364 Asp Gly Leu Tyr Phe Val Ser Phe Ser Arg Asn His Gin Asp Leu Val 60 65 70 GTT TAT AGA CCA ACA TGG CTG ACA TTT TCC TGC AAA GAA GAA GAT TGT 412 Val Tyr Arg Pro Thr Trp Leu Thr Phe Ser Cys Lys Glu Glu Asp Cys 75 80 85 GAT ACT CAT GAT CTT CCT TTG AAA GCT AGA AAG TTT GAG AGC TTC TTC 460 Asp Thr His Asp Leu Pro Leu Lys Ala Arg Lys Phe Glu Ser Phe Phe 90 95 100 ACA CAG TTG TAC AGT GTT ACT CTT GCT TCT AGT GGG GAA CTT ATA TGC 508 Thr Gin Leu Tyr Ser Val Thr Leu Ala Ser Ser Gly Glu Leu lie Cys 105 110 115 120 AAA GAT TTC TTT CTC TAT ATG GAG AGC AAC CAA TTT GGA CTC TTT GCA 556 Lys Asp Phe Phe Leu Tyr Met Glu Ser Asn Gin Phe Gly Leu Phe Ala 125 130 135 ACT TCA ACT GCA CAA ATT CAT GAT GCA CCT CCT ACT GGA GGG GCA ATT 604 Thr Ser Thr Ala Gin lie His Asp Ala Pro Pro Thr Gly Gly Ala lie 140 145 150 CAG GGA GTC CCT TCA GTT GAA AAA ATA ACT TTC CAC CTT TTG AGG TTG 652 Gin Gly Val Pro Ser Val Glu Lys lie Thr Phe His Leu Leu Arg Leu 155 160 165 GTG GAT GGA GCT ATA CTT GAC GAA AGG GTT TTC CAC AAT GAT TAT GTT 700 Val Asp Gly Ala lie Leu Asp Glu Arg Val Phe His Asn Asp Tyr Val 170 175 180 AAT TTG GCA CAT AGC ATT GGT GCT TTC TTG TAT GAT GAT TTG CTT GCT 748 Asn Leu Ala His Ser lie Gly Ala Phe Leu Tyr Asp Asp Leu Leu Ala 185 190 195 200 ATA GTG TCT CTT CGT TAT CAA AGA ATA CAC ATC CTT CAG ATC AGA GAT 796 lie Val Ser Leu Arg Tyr Gin Arg lie His lie Leu Gin lie Arg Asp 205 210 215 TCT GGA GAT CTT GTT GAT GTA CGA GCA ATT GGG GAA TTC TGC CGT GAA 844 Ser Gly Asp Leu Val Asp Val Arg Ala lie Gly Glu Phe Cys Arg Glu 220 225 230 GAT GAT GAA CTT TTT CTC AAT TCC AAT TCC CAG GTG CTT GTA AAT CAT 892 Asp Asp Glu Leu Phe Leu Asn Ser Asn Ser Gin Val Leu Val Asn His 235 240 245 GTT GGA AAT GGT TTT CAT CAT AGT CTG CCT CAA TCA GAA ACT TCT TTC 940 Val Gly Asn Gly Phe His His Ser Leu Pro Gin Ser Glu Thr Ser Phe 250 255 260 CTG AGC GGT ATA AAG CAA CGG CTG CTT TCA TAT ATA TTT CGA GGT ATA 988 Leu Ser Gly lie Lys Gin Arg Leu Leu Ser Tyr lie Phe Arg Gly lie 265 270 275 280 TGG AAT GAA GCT GAC CAA ACC ATG AGA GTG CAG TGC CTG AAG AAG AAG 1036 Trp Asn Glu Ala Asp Gin Thr Met Arg Val Gin Cys Leu Lys Lys Lys 285 290 295 TTT TAC TTC CAC TTT CAA GAT TAC ATT GAC TTG ATT ATC TGG AAG GTG 1084 Phe Tyr Phe His Phe Gin Asp Tyr lie Asp Leu lie lie Trp Lys Val 300 305 310 CAG TTT TTG GAC CGA CAT CAC CTG TTG ATC AAG TTT GGC AGT GTT GAT 1132 Gin Phe Leu Asp Arg His His Leu Leu lie Lys Phe Gly Ser Val Asp 315 320 325 GGT GGG GTA TCC CGA AAT GCT GAC ATC CAT CCT TCT TTT TTT GCT GTT 1180 Gly Gly Val Ser Arg Asn Ala Asp lie His Pro Ser Phe Phe Ala Val 330 335 340 TAC AAT ATG GAG ACT ACT GAA ATT GTT GCA TTT TAT CAG AAC TCA GCC 1228 Tyr Asn Met Glu Thr Thr Glu lie Val Ala Phe Tyr Gin Asn Ser Ala 345 350 355 360 GAT GAG CTT TAT TTC TTG TTC GAG CTG TTC AGC GAC CAT TTT CAC GTT 1276 Asp Glu Leu Tyr Phe Leu Phe Glu Leu Phe Ser Asp His Phe His Val 365 370 375 TCA TCC AAA AGT TCA TTA CAT ATG AAC TTC ATG TCC TCA CAC TCA AAC 1324 Ser Ser Lys Ser Ser Leu His Met Asn Phe Met Ser Ser His Ser Asn 380 385 390 AAC ATC CAC GCC CTC GAG CAA CTA AGG TGT ACA AAG AAC AAA GCA ACC 1372 Asn lie His Ala Leu Glu Gin Leu Arg Cys Thr Lys Asn Lys Ala Thr 395 400 405 AAT TTC TCT CAA TTT GTT AAG AAA ATG ATG GCT TCC TTG CCT TGT AGT 1420 Asn Phe Ser Gin Phe Val Lys Lys Met Met Ala Ser Leu Pro Cys Ser 410 415 420 TGT CAG TCT CAG AGT CCT TCC CCA TAT TTT GAC CAA TCT CTC TTC AGG 1468 Cys Gin Ser Gin Ser Pro Ser Pro Tyr Phe Asp Gin Ser Leu Phe Arg 425 430 435 440 TTT GAC GAG AAG CTT ATT TCA GCT ATT GAC CGC CAT AGA CAG TCT ACT 1516 Phe Asp Glu Lys Leu lie Ser Ala lie Asp Arg His Arg Gin Ser Thr 445 450 455 GAC CAT CCA ATC AAA TTC ATT TCT AGA AGA CAA CCC AAT ATC CTG AAA 1564 Asp His Pro lie Lys Phe lie Ser Arg Arg Gin Pro Asn lie Leu Lys 460 465 470 TTC AAA ATG AAG CCA GGA CCT GAA GCT GGC AGC ACA GAT GGG CGA ACT 1612 Phe Lys Met Lys Pro Gly Pro Glu Ala Gly Ser Thr Asp Gly Arg Thr 475 480 485 AAG AAG ATC TGT TCC TTC CTC TTC CAC CCA ATA TTA CCC CTT GCA CTT 1660 Lys Lys lie Cys Ser Phe Leu Phe His Pro lie Leu Pro Leu Ala Leu 490 495 500 TCT GTT CAA CAA ACC TTG TTT CTG CAG GCA TCA GTT GTA AAT ATC CAT 1708 Ser Val Gin Gin Thr Leu Phe Leu Gin Ala Ser Val Val Asn lie His 505 510 515 520 TTT CGT CGA TAA TGTAAAAACT TAATTTATAT GTTACCGATT TGTTTATAAA 1760 Phe Arg Arg * TTTCTCTAAT AACCTCTAGA TTGAAATCAA CCTAGAAATC ACAAATTCAT CATAACAGAC 1820 CCGTAGATGC TAGTGTCTTT GACTTCTACA TTTTCTTTGT TACAAGAATC AAACAAATGC 1880 TTGATAGAGT GCCAAGACGG TTAGTATGGG TATAAGGATT AGTTCTTCTG TAAGTTTTTG 1940 TTACAGCTTC TCTTCTAATT AATTGATGTA CATTCAGATG TTAAAAAAAA AAAAAAAAAA 2000 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 524 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Met Phe Lys Thr Asn Asn Val Thr Ala Arg Leu Phe Glu Arg Gin lie 1 5 10 15 Cys Thr Pro Ala Pro Gly Thr Ser lie His Arg Ala Arg Arg Phe Tyr 20 25 30 Glu Asn Val Val Pro Ser Tyr Thr lie Tyr Asp Val Glu Cys Pro Asp 35 40 45 His Ser Phe Arg Lys Phe Thr Asp Asp Gly Leu Tyr Phe Val Ser Phe 50 55 60 Ser Arg Asn His Gin Asp Leu Val Val Tyr Arg Pro Thr Trp Leu Thr 65 70 75 80 Phe Ser Cys Lys Glu Glu Asp Cys Asp Thr His Asp Leu Pro Leu Lys 85 90 95 Ala Arg Lys Phe Glu Ser Phe Phe Thr Gin Leu Tyr Ser Val Thr Leu 100 105 110 Ala Ser Ser Gly Glu Leu lie Cys Lys Asp Phe Phe Leu Tyr Met Glu 115 120 125 Ser Asn Gin Phe Gly Leu Phe Ala Thr Ser Thr Ala Gin lie His Asp 130 135 140 Ala Pro Pro Thr Gly Gly Ala lie Gin Gly Val Pro Ser Val Glu Lys 145 150 155 160 lie Thr Phe His Leu Leu Arg Leu Val Asp Gly Ala lie Leu Asp Glu 165 170 175 Arg Val Phe His Asn Asp Tyr Val Asn Leu Ala His Ser lie Gly Ala 180 185 190 Phe Leu Tyr Asp Asp Leu Leu Ala lie Val Ser Leu Arg Tyr Gin Arg 195 200 205 lie His lie Leu Gin lie Arg Asp Ser Gly Asp Leu Val Asp Val Arg 210 215 220 Ala lie Gly Glu Phe Cys Arg Glu Asp Asp Glu Leu Phe Leu Asn Ser 225 230 235 240 Asn Ser Gin Val Leu Val Asn His Val Gly Asn Gly Phe His His Ser 245 250 255 Leu Pro Gin Ser Glu Thr Ser Phe Leu Ser Gly lie Lys Gin Arg Leu 260 265 270 Leu Ser Tyr lie Phe Arg Gly lie Trp Asn Glu Ala Asp Gin Thr Met 275 280 285 Arg Val Gin Cys Leu Lys Lys Lys Phe Tyr Phe His Phe Gin Asp Tyr 290 295 300 lie Asp Leu lie lie Trp Lys Val Gin Phe Leu Asp Arg His His Leu 305 310 315 320 Leu lie Lys Phe Gly Ser Val Asp Gly Gly Val Ser Arg Asn Ala Asp 325 330 335 lie His Pro Ser Phe Phe Ala Val Tyr Asn Met Glu Thr Thr Glu lie 340 345 350 Val Ala Phe Tyr Gin Asn Ser Ala Asp Glu Leu Tyr Phe Leu Phe Glu 355 360 365 Leu Phe Ser Asp His Phe His Val Ser Ser Lys Ser Ser Leu His Met 370 375 380 Asn Phe Met Ser Ser His Ser Asn Asn lie His Ala Leu Glu Gin Leu 385 390 395 400 Arg Cys Thr Lys Asn Lys Ala Thr Asn Phe Ser Gin Phe Val Lys Lys 405 410 415 Met Met Ala Ser Leu Pro Cys Ser Cys Gin Ser Gin Ser Pro Ser Pro 420 425 430 Tyr Phe Asp Gin Ser Leu Phe Arg Phe Asp Glu Lys Leu lie Ser Ala 435 440 445 lie Asp Arg His Arg Gin Ser Thr Asp His Pro lie Lys Phe lie Ser 450 455 460 Arg Arg Gin Pro Asn lie Leu Lys Phe Lys Met Lys Pro Gly Pro Glu 465 470 475 480 Ala Gly Ser Thr Asp Gly Arg Thr Lys Lys lie Cys Ser Phe Leu Phe 485 490 495 His Pro lie Leu Pro Leu Ala Leu Ser Val Gin Gin Thr Leu Phe Leu 500 505 510 Gin Ala Ser Val Val Asn lie His Phe Arg Arg * 515 520 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CATCAACACT GCCAAAC 17 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GAAAGCAGCC GTTGCT 16 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: AGTTCATCAT CTTCACGGC 19 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: GTATGATTCA CTAGTTTAAT GCTGCTGAAA G 31 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CCCATACTAG TCGTCTTGGC ACTCTATCAA G 31 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: GAAGGTAATT TTATATTAAA CATAGAATAG A 31 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GTGATTTCTA GGTTGATTTC AATCTAGA 28 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CAAATCGGTA ACATAT 16 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GGGGAAGCTT TTTCACGAAA TCGGCCCTTA 30 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CCCGGATCCT TCTTTTGCAC TGTGAATGAT TAG 33

Claims: CLAIMS 1. Nucleic acid comprising a nucleotide sequence encoding a protein, or functional portions thereof, which, if altered, is responsible for the light hypersensitive mutant phenotype in Solanum lycopersicum plants, said phenotype comprising a reduced growth of the plant associated with high levels of carotenoids and/or cholophylls and/or flavonoids. 2. Nucleic acid according to claim 1 encoding a tomato protein homologous to at least one of the Arabidopsis COP/DET/FUS family proteins, provided that it is responsible for the light hypersensitive mutant phenotype. 3. Nucleic acid according to claim 2 comprising the nucleotide sequence encoding the TDETI (HP-2) protein, or functional portions thereof. 4. Nucleic acid according to claim 3 comprising a nucleotide sequence encoding the protein having the amino acid sequence of SEQ ID No. 2, or functional portions thereof. 5. Nucleic acid according to claim 4 comprising a nucleotide sequence comprised in SEQ ID No. 1. 6. Nucleic acid according to claim 5 wherein the nucleotide sequence comprised in SEQ ID No. 1 is the sequence from nt. 149 to nt . 1720, or portions thereof. 7. Nucleic acid comprising a nucleotide sequence complementary to SEQ ID No. 1 or portions thereof. 8. Nucleic acid according to claim 3 or 4 comprising a mutation which is able to induce the light hypersensitive phenotype . 9. Nucleic acid according to claim 8 wherein the SEQ ID No. 1 comprises at least a T as a substitute for C at position 1640. 10. Nucleic acid according to claim 8 wherein the SEQ ID No. 1 comprises at least a nucleotide deletion from nt . 1581 to nt. 1589. 11. Expression vector comprising the nucleic acid according to any one of preceding claims under the control of a promoter that is active in plants. 12. Vector according to claim 11 wherein said promoter is selectively active only in a few plant organs. 13. Vector according to claim 12 wherein said promoter is active in the fruits. 14. Vector according to any one of preceding claims 11-13 which is able to control the transcription of an antisense RNA. 15. Vector according to claim 14 wherein said vector is the pBIN-E8-HP2-ASl vector or the pBIN-E8-HP2-ASl vector. 16. Use of vector according to any one of preceding claims 11-15 to produce transgenic plants containing the nucleic acid according to any one of preceding claims 1-10, under the control of specific regulatory sequences, preferably in selected organs of plants. 17. Use of vector according to claim 16 wherein said transgenic plant belongs to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae or ornamental species. 18. Transgenic plant obtainable by the use of transforming vector according to any one of the preceding claims 11-15. 19. Transgenic plant according to claim 18 belonging to pepper, eggplant, soybean, grape, melon, rice, carrot, spinach, citrus, pomaceae and ornamental species. 20. Protein, or portions thereof, which, if altered, is responsible for the light hypersensitive phenotype in Solanum lycopersicum plants. 21. A protein according to claim 20 homologous with at least one of the Arabidopsis COP/DET/FUS family proteins, whose alteration is responsible for a light hypersensitive phenotype . 22. Protein according to claim 21 comprising the amino acid sequence of SEQ ID No. 2, or portions thereof. 23. Protein according to claim 22 comprising an alteration to the amino acid sequence of SEQ ID No. 2, or portions thereof, which are able to induce a light hypersensitive phenotype. 24. Protein according to claim 23 comprising at least an alteration in the SEQ ID No. 1 C-terminal portion. 25. Protein according to claim 24 wherein said alteration in the C-terminal portion comprises a substitution of proline at 498 position. 26. Protein according to claim 25 wherein said substitution comprises a serine as a substitute for proline . 27. Protein according to claim 24 wherein said alteration in the C-terminal portion comprises a deletion of at least one amino acid in the second NLS domain. 28. Protein according to claim 27 wherein said deletion comprises at least three amino acids. 29. Protein according to claim 28 wherein said deletion comprising at least three amino acids is from aa. 478 to aa. 480.

Inventors: BOWLER CHRIS, MUSTILLI ANNA CHIARA, BOWLER, CHRIS, MUSTILLI, ANNA, CHIARA
Applicants: BOWLER CHRIS, MUSTILLI ANNA CHIARA, STAZIONE ZOOL ANTON DOHRN, BOWLER, CHRIS, MUSTILLI, ANNA, CHIARA, STAZIONE ZOOLOGICA "ANTON DOHRN"
Application year: 1998
Main classification: C12N15
IPC codes: A01H5, C07K14, C12N15