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.

That which is claimed: 
     
         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 .