Trichome-specific transcription factor modulating terpene biosynthesis

Abstract

The present invention relates to identification and isolation of zinc finger transcription factor in tomato that specifically expresses in glandular trichomes of Solanum lycopersicum cultivar Moneymaker and binds to the promoters of the genes encoding Terpene Synthase 5 (also known as Monoterpene Synthase1) and Terpene Synthase 11 (also known as Sesquiterpene Synthase 1). The invention provides the isolated, recombinant or synthetic polynucleotides encoding the polypeptide sequences of SEQ ID NO:2 and variants and fragments thereof. The invention also provides constructs, vectors, host cells and plants genetically modified to contain the polynucleotides of the invention. The methods for producing plants with altered levels of terpenes, including transformed and mutant plants, are also provided.

1 . An isolated, synthetic or recombinant nucleic acid sequence selected from the group comprising:
 a) a nucleic acid sequence of SEQ ID NO: 1;   b) a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO:2;   c) a nucleic acid sequence that is at least 60% identical to the nucleic acid sequences of (a) or (b), and encodes a transcription factor that regulates terpene biosynthesis;   d) a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence which has been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO:2 and which polypeptide is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; and   e) a nucleic acid sequence that hybridizes under stringent conditions to the nucleic acid sequences of (a), (b), (c), or (d), or   f) a nucleic acid sequence that hybridizes under stringent conditions to the complement of the nucleic acid sequences of (a), (b), (c), or (d) and encodes a polypeptide capable of regulating terpene biosynthesis in a plant.   
     
     
         2 . A chimeric gene comprising a nucleic acid sequence according to  claim 1 . 
     
     
         3 . A vector comprising a chimeric gene according to  claim 2 . 
     
     
         4 . A host cell comprising a vector according to  claim 3 . 
     
     
         5 . A polypeptide with DNA binding activity that regulates terpene biosynthesis in a plant having an amino acid sequence selected from the group of:
 (a) an amino acid sequence of SEQ ID NO: 2;   (b) an amino acid sequence which has been derived, by way of one or more amino acid substitutions, deletions or insertions, from the polypeptide having the amino acid sequence of SEQ ID NO:2 and which polypeptide is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2; and   (c) an amino acid sequence that is at least 60% identical to the amino acid sequence according to (a).   
     
     
         6 . The polypeptide according to  claim 5  capable of binding a nucleic acid sequence of a promoter that is operably linked to at least one gene involved in terpene biosynthesis in the plant. 
     
     
         7 . The polypeptide according to  claim 6  wherein the gene is selected from the group comprising a Terpene Synthase 5 (TPS5) and a Terpene Synthase 11 (TPS11). 
     
     
         8 . The polypeptide according to  claim 6  wherein the promoter comprises a trichome-specific promoter. 
     
     
         9 . A method for increasing production of at least one terpene in a plant, comprising modifying the plant to have an increased copy number of a nucleic acid sequence of SEQ ID NO:1, or a nucleic acid sequence with at least 60% identity to the nucleic acid sequence of SEQ ID NO: 1, compared to a non-modified plant of the same genetic background, thereby increasing the production of the at least one terpene in the modified plant 
     
     
         10 . A method for increasing production of at least one terpene in a plant comprising:
 (a) contacting a plant cell with a composition comprising a vector comprising a nucleic acid sequence having at least 60% identity to SEQ ID NO:1 or a functional fragment thereof;   (b) selecting the plant cell transformed with the vector wherein the plant cell overexpresses the nucleic acid sequence or the fragment wherein overexpression results in an increased level of the at least one terpene in the cell compared to a non-transformed plant cell; and   (c) regenerating the plant from the transformed cell of (b) wherein the plant has the increased level of the at least one terpene compared to a non-transformed plant of the same genetic background.   
     
     
         11 . A method for increasing production of at least one terpene in a population of plants by selectively breeding the plant of  claim 8  to produce the population having the increased level of the at least one terpene. 
     
     
         12 . A method for reducing production of at least one terpene in a plant, comprising the step of impairing expression of functional TF19(6) protein in a plant via genetic modification, and/or the step of reducing binding of functional TF19(6) protein to the promoter sequence of the nucleic acid sequence coding for Terpene Synthase 5 (TPS5) and/or Terpene Synthase 11 (TPS11). 
     
     
         13 . A method according to  claim 12 , said method comprising the step of modifying the plant to have at least one mutation in a nucleic acid sequence of SEQ ID NO: 1 or a sequence with at least 60% identity to the nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid sequence of SEQ ID NO: 3 or a sequence with at least 60% identity to the nucleic acid sequence of SEQ ID NO: 3, wherein the mutation results in a decrease of expression of the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:2, and/or a loss of function of the amino acid sequence of SEQ ID NO:2 or a variant thereof having at least 60% sequence identity to the amino acid sequence of SEQ ID NO:2 compared to a non-modified plant of the same genetic background, thereby reducing the level of the at least one terpene in the modified plant. 
     
     
         14 . The method according to  claim 13  wherein the mutation comprises a substitution, a deletion, an insertion or an addition of at least one nucleotide. 
     
     
         15 . The method according to  claim 12  wherein the step of impairing expression of functional TF19(6) protein in a plant via genetic modification comprises modifying the plant to have an increased level of RNA having a nucleic acid sequence at least in portion complementary to a nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid sequence with at least 60% identity to the nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid sequence of SEQ ID NO: 3 or a nucleic acid sequence with at least 60% identity to the nucleic acid sequence of SEQ ID NO: 3, compared to a non-modified plant of the same genetic background, thereby decreasing production of the at least one terpene in the modified plant. 
     
     
         16 . A method for reducing terpene levels in a population of plants, comprising:
 (a) providing at least one plants within a population of plants comprising a mutation in a gene having a nucleic acid sequence of SEQ ID NO: 1 or a sequence with at least 60% identity to the nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid sequence of SEQ ID NO: 3 or a sequence with at least 60% identity to the nucleic acid sequence of SEQ ID NO: 3; and   (b) selectively breeding the at least one mutant plant of (a) to produce the population of plants having the reduced terpene levels.   
     
     
         17 . The method according to  claim 9  wherein the terpene comprises at least one of a monoterpene and a sesquiterpene that repel insects. 
     
     
         18 . The method according to  claim 17  wherein the monoterpene comprises at least one compound selected from the group of: linalool, β-myrcene, para-cymene, γ-terpinene, α-terpinene, and α-phellandrene. 
     
     
         19 . The method according to  claim 17  wherein the sesquiterpene comprises at least one compound selected from the group of: neralidol, germacrene, R-curcumine, S-curcumine and 7-epizingiberene. 
     
     
         20 . The method according to  claim 9  wherein the terpene comprises at least one of a monoterpene and a sesquiterpene that attract insects. 
     
     
         21 . The method according to  claim 20  wherein the monoterpene comprises at least one compound selected from the group of: β-phellandrene, limonene and 2-carene. 
     
     
         22 . The method according to  claim 20  wherein the sesquiterpene comprises at least β-caryophyllene. 
     
     
         23 . The method according to  claim 17  wherein the insects comprise sap-sucking insects and blood-sucking insects. 
     
     
         24 . The method according to  claim 23  wherein the sap-sucking insects comprise psyllids, whiteflies, aphids, mealybugs, plant hoppers and scale insects. 
     
     
         25 . The method according to  claim 24 , wherein the sap-sucking insects further comprise thrips, mites and leaf hoppers. 
     
     
         26 . The method according to  claim 23  wherein the blood sucking insects comprise mosquito, ticks and midges. 
     
     
         27 . The method according to  claim 9  wherein the plant is at least one crop plant selected from the group of: tomato, pepper, eggplant, lettuce, oilseed rape, broccoli, cauliflower, cabbage crops, cucumber, melon, pumpkin, squash, peanut, soybeans, corn, cotton, beans, cassava, potatoes, sweet potatoes and okra. 
     
     
         28 . The method according to  claim 27  wherein the plant comprises at least one plant selected from a Solanaceae family. 
     
     
         29 . The method according to  claim 9  wherein the plant is at least one ornamental plant selected from the group of: hibiscus, poinsettia, lilies, iris, rose and petunia. 
     
     
         30 . A plant obtainable by a method according to  claim 9 . 
     
     
         31 . A plant comprising a chimeric gene according to  claim 2 . 
     
     
         32 . The plant according to  claim 30  wherein the plant belongs to the Solanaceae family. 
     
     
         33 . A tissue culture obtained from the plant of  claim 30  wherein the culture has enhanced production or secretion of at least one terpene, terpene isomer, or terpene derivative. 
     
     
         34 . A method for producing a terpene, terpene isomer, or terpene analog comprising isolating the terpene, terpene isomer or terpene analog from the tissue culture of  claim 33 . 
     
     
         35 . A method for a marker-assisted introgression of production of at least one terpene into a plant comprising the steps of:
 (a) identifying a difference in a gene encoding a polypeptide having an amino acid sequence of SEQ ID NO:2 between a plant from the Solanaceae family and a sexually compatible plant wherein the gene with the difference comprises a molecular marker associated with the presence of the at least one terpene in the plant;   (b) making a cross between the plant having the molecular marker and the sexually compatible plant of (a);   (c) screening a progeny resulting from the cross for the presence of the molecular marker;   (d) identifying the plant within the progeny having the molecular marker and thereby identifying the plant producing the at least one terpene.