Reduction of Tobacco-Specific Nitrosamines Using Genetic Modification to Elevate Production of Native Antioxidants in Tobacco

Abstract

Cured tobacco having a lower concentration of tobacco specific nitrosamines, can be produced from a transgenic tobacco plant having a heterologous nucleotide sequence encoding a polypeptide that catalyzes production of active oxygen species. The heterologous nucleotide sequence is operably coupled to a regulatory nucleotide sequence that directs expression of the polypeptide. The regulatory nucleotide sequence can include constitutive and/or inducible promoter elements.

1 . Cured tobacco having lower concentrations of tobacco specific nitrosamines, which is produced from a transgenic tobacco plant comprising a heterologous nucleotide sequence encoding a polypeptide that catalyzes production of active oxygen species, wherein the heterologous nucleotide sequence is operably coupled to a regulatory nucleotide sequence that directs expression of the polypeptide. 
     
     
         2 . The tobacco of  claim 1 , wherein the polypeptide that catalyzes production of active oxygen species is an oxidase. 
     
     
         3 . The tobacco of  claim 2 , wherein the polypeptide that catalyzes production of active oxygen species is an oxidase selected from among oxalate oxidase and glucose oxidase. 
     
     
         4 . The tobacco of  claim 1 , wherein the regulatory nucleotide sequence comprises a constitutively active promoter sequence. 
     
     
         5 . The tobacco of  claim 1 , wherein the regulatory nucleotide sequence comprises an inducible promoter sequence. 
     
     
         6 . The tobacco of  claim 1 , wherein the regulatory nucleotide sequence comprises a promoter sequence that is induced by plant wounding, heat, light, drought, or a combination thereof. 
     
     
         7 . The tobacco of  claim 5 , wherein the promoter is induced by a natural signal associated with senescence. 
     
     
         8 . The tobacco of  claim 5 , wherein the promoter is inducible by a synthetic chemical. 
     
     
         9 . The tobacco of  claim 1 , wherein the regulatory nucleotide sequence comprises a promoter sequence that is most active in root and/or stem tissue. 
     
     
         10 - 12 . (canceled) 
     
     
         13 . A method of producing cured tobacco having reduced concentrations of tobacco-specific nitrosamines, the method comprising:
 growing a transgenic tobacco plant comprising a heterologous nucleotide sequence encoding a polypeptide that catalyzes production of active oxygen species, wherein the heterologous nucleotide sequence is operably coupled to a regulatory nucleotide sequence that directs expression of the polypeptide;   harvesting the tobacco; and   curing the tobacco.   
     
     
         14 . The method of  claim 13 , wherein the polypeptide that catalyzes production of active oxygen species is an oxidase. 
     
     
         15 . The method of  claim 13 , wherein the polypeptide that catalyzes production of active oxygen species is an oxidase selected from among oxalate oxidase and glucose oxidase. 
     
     
         16 . The method of  claim 13 , wherein the regulatory nucleotide sequence comprises a constitutively active promoter sequence. 
     
     
         17 . The method of  claim 13 , wherein the regulatory nucleotide sequence comprises an inducible promoter. 
     
     
         18 . The method of  claim 17 , comprising causing induction of the promoter within the period about 3 weeks prior to harvesting the tobacco to about 48 hours after harvesting the tobacco. 
     
     
         19 . The method of  claim 17 , wherein the regulatory nucleotide sequence comprises a promoter sequence that is induced by chemical treatment, plant wounding, heat, light, drought, or a combination thereof. 
     
     
         20 . The method of  claim 17 , comprising causing induction of the promoter about 1 to 3 weeks prior to harvesting the tobacco. 
     
     
         21 . The method of  claim 17 , wherein the promoter is induced by one or more natural signals associated with senescence. 
     
     
         22 . The method of  claim 19 , further comprising root pruning and/or severing the xylem tissue of the tobacco plant prior to harvesting. 
     
     
         23 . The method of  claim 19 , wherein the promoter is inducible by chemical treatment and further comprising applying an inducer by spraying or irrigation. 
     
     
         24 - 29 . (canceled)