Cytokinin Oxidase-Like Sequences and Methods of Use

Abstract

Methods and compositions for modulating plant development are provided. Polynucleotide sequences and amino acid sequences encoding cytokinin oxidase polypeptides are provided. The sequences can be used in a variety of methods including modulating root development, modulating floral development, modulating leaf and/or shoot development, modulating seed size and/or weight, modulating tolerance under abiotic stress, and modulating resistance to pathogens. Polynucleotides comprising CKX promoters are also provided. The promoters can be used to regulate expression of a sequence of interest. Transformed plants, plant cells, tissues, and seed are also provided.

1 . An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
 (a) an amino acid sequence comprising SEQ ID NO: 3, 6, 9, 12, 53, 59 or 62;   (b) an amino acid sequence comprising at least 85% sequence identity to the full length of SEQ ID NO: 3, 6, 9, 12, 53, 59 or 62, wherein said polypeptide has cytokinin oxidase activity;   (c) an amino acid sequence encoded by a nucleotide sequence that hybridizes under stringent conditions to the complement of SEQ ID NO: 2, 5, 8, 11, 52, 58 or 61, wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C.; and,   (d) an amino acid sequence comprising a fragment of SEQ ID NO: 3, 6, 9, 12, 53, 59 or 62, wherein said polypeptide retains cytokinin oxidase activity.   
     
     
         2 . An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence comprising SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 57, 58, 60 or 61;   (b) a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 3, 6, 9, 12, 53, 59 or 62;   (c) a nucleotide sequence comprising at least 85% sequence identity to SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 57, 58, 60 or 61, or to the coding sequence thereof, wherein said polynucleotide encodes a polypeptide having cytokinin oxidase activity;   (d) a nucleotide sequence comprising at least 50 consecutive nucleotides of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 57, 58, 60 or 61, or a complement thereof; and,   (e) a nucleotide sequence that hybridizes under stringent conditions to the complement of the nucleotide sequence of a), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C.   
     
     
         3 . An expression cassette comprising the polynucleotide of  claim 2  operably linked to a promoter that drives expression in a plant. 
     
     
         4 . A plant comprising the expression cassette of  claim 3 . 
     
     
         5 . The plant of  claim 4 , wherein said plant has a modulated cytokinin level when compared to a control plant. 
     
     
         6 . The expression cassette of  claim 3 , wherein said promoter is a tissue-preferred promoter, a constitutive promoter, or an inducible promoter. 
     
     
         7 . The plant of  claim 4 , wherein said plant, when compared to a control plant, displays one or more traits selected from the group consisting of: modulated floral development, modulated root development, an altered shoot-to-root ratio, improved plant vigor, and increased plant biomass. 
     
     
         8 . The plant of  claim 4 , wherein said plant has increased average seed size or increased total seed weight, or both, when compared to a control plant. 
     
     
         9 . The plant of  claim 4 , wherein the stress tolerance of said plant is maintained or improved when compared to a control plant. 
     
     
         10 . The plant of  claim 9 , wherein said plant is  Zea mays  and tip kernel abortion is reduced. 
     
     
         11 . A transformed seed of the plant of  claim 4 . 
     
     
         12 . A plant that is genetically modified to affect expression of a native genomic locus, said genomic locus encoding a polypeptide selected from the group consisting of:
 (a) an amino acid sequence comprising SEQ ID NO: 3, 6, 9, 12, 53, 59 or 62; and   (b) an amino acid sequence comprising at least 85% sequence identity to the entire length of SEQ ID NO: 6, 9, 12, 53, 59 or 62, wherein said polypeptide has cytokinin oxidase activity;   wherein said plant is genetically modified to reduce the level or activity of said polypeptide.   
     
     
         13 . A method for modulating the level or activity of cytokinin oxidase in a plant or plant part, comprising introducing into said plant a polynucleotide comprising a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence comprising SEQ ID NO: 2, 5, 8, 11, 52, 58 or 61;   (b) a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 3, 6, 9, 12, 53, 59 or 62;   (c) a nucleotide sequence having at least 85% sequence identity to the full length of SEQ ID NO: 2, 5, 8, 11, 52, 58 or 61, wherein said polynucleotide encodes a polypeptide having cytokinin oxidase activity; and   (d) a nucleotide sequence that hybridizes under stringent conditions to the complement of the polynucleotide of a), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C., and wherein said polynucleotide encodes a polypeptide having cytokinin oxidase activity.   
     
     
         14 . The method of  claim 13 , wherein said polynucleotide is operably linked to a tissue-specific promoter, a constitutive promoter, or an inducible promoter. 
     
     
         15 . The method of  claim 13 , wherein root development of the plant is modulated. 
     
     
         16 . The method of  claim 14 , wherein the promoter drives root-preferred expression of the operably-linked polynucleotide. 
     
     
         17 . The method of  claim 16 , wherein the promoter is the NAS2 promoter or the ROOTMET2 promoter. 
     
     
         18 . The method of  claim 17 , wherein the promoter is operably linked to a polynucleotide encoding a polypeptide at least 85% identical to SEQ ID NO: 2. 
     
     
         19 . A method for reducing the level or activity of cytokinin oxidase in a plant or plant part, comprising introducing into said plant a polynucleotide comprising a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence fragment comprising at least 50 consecutive nucleotides of SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 51, 52, 57, 58, 61, 62 or a complement thereof;   (b) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of part (a), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C.;   (c) a nucleotide sequence fragment comprising at least 50 consecutive nucleotides of SEQ ID NO: 17, 18, 13, 14, 15, 16, 69, 70 or 63; and   (d) a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of part (c), wherein said stringent conditions comprise hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60° C. to 65° C.   
     
     
         20 . The method of  claim 19 , wherein said plant has increased average seed size or increased total seed weight, or both, when compared to a control plant. 
     
     
         21 . The method of  claim 19 , wherein said plant is maize, wheat, rice, barley, sorghum, or rye. 
     
     
         22 . The method of  claim 19 , wherein the stress tolerance of said plant is maintained or improved when compared to a control plant. 
     
     
         23 . The method of  claim 22 , wherein the plant is  Zea mays  and tip kernel abortion is reduced relative to a control. 
     
     
         24 . The method of  claim 19 , wherein said polynucleotide is configured within a hairpin construct. 
     
     
         25 . A method of suppressing cytokinin oxidase activity in a plant, comprising transformation of a plant host cell with the genetic construct of  claim 24  and regenerating from said transformed cell a transgenic plant wherein expression of one or more endogenous cytokinin oxidase genes is reduced or eliminated. 
     
     
         26 . A first polynucleotide comprising a nucleotide sequence comprising SEQ ID NO: 13, 14, 15, 16, 63, 69 or 70, wherein said polynucleotide drives expression of a second, operably-linked, polynucleotide. 
     
     
         27 . A DNA construct comprising a promoter operably linked to a nucleotide sequence of interest, wherein said promoter comprises the polynucleotide of  claim 26  or a functional fragment thereof.