Nucleotide sequences encoding the tomato light...

Details Nucleotide sequences encoding the tomato light... Nucleotide sequences encoding the tomato light...

2000-08-03

2002-08-06

Fredman, Jeffrey (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S022100, C536S024300, C536S024500, C435S006120, C435S091200, C435S320100, C800S290000

Reexamination Certificate

active

06429299

ABSTRACT:

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 phytochromes 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 aurea (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 photomorphogenic 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; Whitelam 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 Chua, 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, atv, 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-1
w
(Peters et al., 1989) and hp-2
j
(Van 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 DET1 gene, which belongs to the above described constitutive COP/DET/FUS mutant group. Therefore the tomato HP-2 gene has been renamed TDET1.
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 mutatio

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