Nucleic acid sequences encoding transcription factors regulating alkaloid biosynthesis and their use in modifying plant metabolism

1. A method for reducing a nicotinic alkaloid in a Nicotiana plant, comprising down-regulating a transcription factor that positively regulates nicotinic alkaloid biosynthesis, wherein the transcription factor is down-regulated by:

(a) introducing into a Nicotiana plant cell a nucleic acid comprising at least 21 consecutive nucleotides of a cDNA molecule comprising a nucleotide sequence selected from the group consisting of:

(i) a nucleotide sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12;

(ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 13;

(iii) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (i) or (ii), and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis; and

(iv) a nucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequences of (i), (ii), and/or (iii), and which encodes a transcription factor that regulates nicotinic alkaloid biosynthesis, wherein the high stringency conditions comprise hybridization at 68° C., washing with 2×SSE and 0.05% SDS five times at 68° C., and washing with 0.1×SSC and 0.1% SDS at 68° C. for 1 hour;

wherein the consecutive nucleotides are in sense orientation, antisense orientation, or both;

(b) producing a Nicotiana plant comprising the Nicotiana plant cell; and

(c) growing the Nicotiana plant under conditions whereby the nucleotide sequence decreases levels of the transcription factor in the Nicotiana plant as compared to a control Nicotiana plant grown under similar conditions.

2. The method of claim 1 , further comprising suppressing within the Nicotiana plant at least one of NBB1, A622, quinolate phospho-ribosyltransferase (QPT), putrescine-N-methyltransferase (PMT), and N-methylputrescine oxidase (MPO).

3. The method of claim 1 , further comprising suppressing the expression of at least one additional transcription factor that positively regulates nicotinic alkaloid biosynthesis.

4. The method of claim 1 , further comprising overexpressing at least one transcription factor that negatively regulates nicotinic alkaloid biosynthesis.

5. A method for reducing a nicotinic alkaloid in a Nicotiana plant, comprising down-regulating a transcription factor that positively regulates nicotinic alkaloid biosynthesis, wherein the transcription factor is down-regulated by:

(a) introducing into a population of Nicotiana plant cells a reagent for site-directed mutagenesis of a target comprising at least 21 consecutive nucleotides of a cDNA molecule comprising a nucleotide sequence selected from the group consisting of:

(i) a nucleotide sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12;

(ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 13;

(iii) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (i) or (ii), and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis; and

(iv) a nucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequences of (i), (ii), and/or (iii), and which encodes a transcription factor that regulates nicotinic alkaloid biosynthesis, wherein the high stringency conditions comprise hybridization at 68° C., washing with 2×SSE and 0.05% SDS five times at 68° C., and washing with 0.1×SSC and 0.1% SDS at 68° C. for 1 hour; and

(b) detecting and selecting a target mutated Nicotiana plant cell or a Nicotiana plant derived from such a Nicotiana cell, wherein the target mutated Nicotiana plant cell or Nicotiana plant has a mutation in a gene encoding the transcription factor positively regulating nicotinic alkaloid biosynthesis and reduced nicotinic alkaloid content as compared to a control Nicotiana plant.

6. The method of claim 5 , wherein the reagent is a recombinagenic oligonucleobase.

7. The method of claim 5 , wherein the reagent is a targeted nuclease.

8. A mutated Nicotiana plant produced by the method of claim 5 , wherein the Nicotiana plant has reduced expression of a transcription factor that positively regulates nicotinic alkaloid biosynthesis and reduced nicotinic alkaloid content, as compared to a control Nicotiana plant.

9. A product comprising the mutated Nicotiana plant of claim 8 or portions thereof, wherein the plant or portions thereof comprise the mutation in the gene encoding the transcription factor positively regulating nicotinic alkaloid biosynthesis, and wherein the product has a reduced level of a nicotinic alkaloid as compared to a product produced from a control Nicotiana plant.

10. Seeds from the mutated Nicotiana plant of claim 8 , wherein the seeds comprise the mutation.

11. A method for reducing nicotinic alkaloid levels in a population of Nicotiana plants, comprising:

(a) providing a population of mutated Nicotiana plants;

(b) detecting and selecting a target mutated Nicotiana plant within the population, wherein the target mutated Nicotiana plant has decreased expression of a transcription factor that positively regulates nicotinic alkaloid biosynthesis as compared to a control Nicotiana plant, wherein the detection comprises using a cDNA molecule as a primer or a probe, and wherein the cDNA molecule comprises a nucleotide sequence selected from the group consisting of:

(i) a nucleotide sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12;

(ii) a nucleotide sequence that encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 13;

(iii) a nucleotide sequence that is at least 90% identical to the nucleotide sequences of (i) or (ii), and which encodes a transcription factor that positively regulates nicotinic alkaloid biosynthesis; and

(iv) a nucleotide sequence that hybridizes under high stringency conditions to the nucleotide sequences of (i), (ii), and/or (iii), and which encodes a transcription factor that regulates nicotinic alkaloid biosynthesis, wherein the high stringency conditions comprise hybridization at 68° C., washing with 2×SSE and 0.05% SDS five times at 68° C., and washing with 0.1×SSC and 0.1% SDS at 68° C. for 1 hour; and

(c) selectively breeding the target mutated Nicotiana plant to produce a population of Nicotiana plants having decreased expression of a transcription factor that positively regulates nicotinic alkaloid biosynthesis as compared to a population of control Nicotiana plants.

12. A mutated nicotinic alkaloid-producing Nicotiana plant produced by the method of claim 11 , wherein the Nicotiana plant has reduced expression of a transcription factor that positively regulates nicotinic alkaloid biosynthesis and reduced nicotinic alkaloid content, as compared to a control Nicotiana plant.

13. The mutated Nicotiana plant of claim 12 , wherein the Nicotiana plant is a Nicotiana tabacum plant.

14. A product comprising the mutated Nicotiana plant of claim 12 or portions thereof, wherein the plant or portions thereof comprise a mutation in the transcription factor that positively regulates nicotinic alkaloid biosynthesis, and wherein the product has a reduced level of a nicotinic alkaloid as compared to a product produced from a control Nicotiana plant.

15. Seeds from the mutated Nicotiana plant of claim 12 , wherein the seeds comprise the mutation.