Methods and composition for modulating flavonoid content

Abstract

A method for manipulating the production of flavonoids in tomatoes by manipulating gene activity in the flavonoid biosynthetic pathway by expressing genes encoding chalcone isomerase, compositions for use in such a method and tomato plants having altered flavonoid levels are disclosed.

1 . A method for producing a plant capable of exhibiting altered levels of flavonoids comprising incorporating into said plant one or more gene sequences encoding a protein with chalcone isomerase activity, or incorporating a nucleotide sequence encoding a protein functionally equivalent thereto.  
     
     
         2 . Method according to  claim 1  characterised in that said gene or genes encode chalcone isomerase, isolated from a species selected from the group comprising tomato plant, petunia, maize, arabidopsis, alfalfa, pea, bean, grape, apple.  
     
     
         3 . Method according to  claim 1  characterised in that said gene or genes or nucleotide sequence encode the protein chalcone isomerase, isolated from petunia.  
     
     
         4 . A method according to  claim 1  characterised in that said plant is a tomato plant.  
     
     
         5 . A method according to any one of  claims 1  to  4  characterised in that levels of “specific flavonoids” in said plant are increased compared to untransformed plants.  
     
     
         6 . A method according to any one of claims  1 - 5  characterised in that the level of “specific flavonoids” in transformed plants is at least 4 times higher than in similar untransformed plants, more preferred 5-100, most preferred 10-40 times higher than in similar untransformed plants.  
     
     
         7 . A method according to any one of  claims 1  to  6  characterised in that the level of specific flavonoids in the peel of the fruit of said plant is increased.  
     
     
         8 . A method according to any one of  claims 1  to  7  wherein said flavonoid is a flavonol.  
     
     
         9 . A method according to any of  claims 1  to  7  characterised in that the flavonoid is quercetin or kaempferol or their glycosides or any other derivative thereof.  
     
     
         10 . A method according to any one of  claims 1  to  8  characterised in that the introduced gene comprises a nucleotide sequence or complementary nucleotide sequence selected from: 
 (i) a nucleotide sequence, encoding an amino acid sequence having at least 40% similarity, to seq ID No1;  
 (ii) a nucleotide sequence capable of hybridising under low stringent conditions to a sequence selected from the group of sequences set forth under (i) above;  
 (iii) a nucleotide sequence encoding a protein that is being functionally equivalent to the protein encoded by seq ID no 1.  
 
     
     
         11 . A method according to  claim 10 , characterised in that said gene comprises a nucleotide sequence encoding an amino acid sequence having at least 60% similarity, preferably at least 90%, more preferred at least 95%, most preferred at least 95% similarity to the sequence as set forth in seq ID No1.  
     
     
         12 . A method according to  claim 10 , characterised in that said gene comprises a nucleotide sequence, encoding an amino acid sequence having 99-100% similarity, to seq ID No1.  
     
     
         13 . A method according to any of claims  1 - 12 , characterised in that said nucleotide sequence comprises a sequence which has at least 50, more preferably at least 60% similarity, more preferred at least 75%, even more preferred at least 80%, still more preferred at least 90%, most preferred 95-100% similarity to seq ID no 2, and whereby said sequence encodes a protein having chalcone isomerase activity.  
     
     
         14 . A method according to any one of claims  1 - 13  characterised in that the gene encoding a protein with chalcone isomerase is operably linked to a promoter.  
     
     
         15 . A method according to  claim 14  characterised in that the promoter is selected from the group of: 
 (a) constitutive promoters, such as carnation edged ring virus, cauliflower mosaic virus 35 S promoter, enhanced cauliflower mosaic virus 35 S promoter;  
 (b) fruit specific promoters;  
 (c) any other suitable promoter such as GBSS (granular bound starch synthase) promoter.  
 
     
     
         16 . A method according to  claim 15  characterised in that the promoter is a fruit specific promoter selected from the group of PG, 2A11, E8, E4, and fpb11.  
     
     
         17 . A method according to any of claims  1 - 16  comprising the additional step increasing phenylalanine biosynthesis.  
     
     
         18 . A plant having one or more transgenes, each encoding a protein with chalcone isomerase activity or a protein functionally equivalent thereto, incorporated into its genome such that its ability to produce flavonoids is altered.  
     
     
         19 . A plant according to  claim 18 , whereby said plant is a tomato plant, prepared according to the method of any one of  claims 1  to  17 .  
     
     
         20 . A DNA construct suitable for use to overexpress the encoded protein, comprising sequences coding for a protein with chalcone isomerase activity, or a functionally equivalent sequence thereof, operably linked to a promoter.  
     
     
         21 . A plant comprising a DNA construct according to  claim 20 .  
     
     
         22 . A transformed plant having enhanced levels of specific flavonoids compared to similar, untransformed plants.  
     
     
         23 . A tomato plant having enhanced levels of specific flavonoids in the peel of the fruit compared to the level of flavonoids in the peel of the fruit of untransformed plants.  
     
     
         24 . Seeds, fruits, progeny and hybrids of a plant according to any one of claims  18 ,  19 ,  21 ,  22 , or  23 .  
     
     
         25 . A food product comprising at least part of a plant according to any one of claims  18 ,  19 ,  21 ,  22 , or  23 .  
     
     
         26 . A food product according to  claim 25  characterised in that the food product is sauce, dressing, ketchup or soup.  
     
     
         27 . A skin or hair protective product comprising at least part of a plant according to any one of claims  18 ,  19 ,  21 ,  22 , or  23 .  
     
     
         28 . A pharmaceutical product comprising at least part of a plant according to any one of claims  18 ,  19 ,  21 ,  22 , or  23 .