Isopentenyl transferase sequences and methods of use

Abstract

Methods and compositions for modulating plant development are provided. Polynucleotide sequences and amino acid sequences encoding isopentenyl transferase (IPT) 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 senescence, modulating seed size and/or weight, and modulating tolerance of plants to abiotic stress. Polynucleotides comprising an IPT promoter are also provided. The promoter can be used to regulate expression of a sequence of interest. Transformed plants, plant cell, tissues, and seed are also provided.

1 . An isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, or 77.  
     
     
         2 . An isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, or 66.  
     
     
         3 . An isolated polypeptide comprising an amino acid sequence selected from the group consisting of: 
 (a) an amino acid sequence comprising at least 85% sequence identity to SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66, or 77, wherein said polypeptide has cytokinin synthesis activity;    (b) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide represented by SEQ ID NO: 1, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74, or 76, 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., wherein said polypeptide retains cytokinin synthesis activity; and,    (d) an amino acid sequence comprising at least 50 consecutive amino acids of SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66, or 77, wherein said polypeptide retains cytokinin synthesis activity.    
     
     
         3 . An isolated polynucleotide comprising a nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, or 76.  
     
     
         4 . An isolated polynucleotide comprising a nucleotide sequence of SEQ ID NO: 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, or 74.  
     
     
         5 . An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: 
 (a) a nucleotide sequence encoding an amino acid sequence comprising SEQ ID NO: 2, 6, 9, 12, 15, 18, 23, 27, 41, 43, 46, 49, 52, 54, 57, 59, 61, 63, 66, or 77;    (b) a nucleotide sequence comprising at least 85% sequence identity to SEQ ID NO: 1, 3, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74, or 76, wherein said polynucleotide encodes a polypeptide having cytokinin synthesis activity;    (c) a nucleotide sequence comprising at least 50 consecutive nucleotides of SEQ ID NO: 1, 3, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 21, 22, 24, 26, 28, 40, 42, 44, 45, 47, 48, 50, 51, 53, 55, 56, 58, 60, 62, 64, 65, 69, 70, 71, 72, 73, 74, or 76, wherein said polynucleotide encodes a polypeptide having cytokinin synthesis activity; and,    (d) a nucleotide sequence which represents a polynucleotide that hybridizes under stringent conditions to the complement of a polynucleotide represented by 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.    
     
     
         6 . A transgenic plant comprising a polynucleotide operably linked to a promoter that drives expression in the plant, wherein said polynucleotide comprises a nucleotide sequence of  claim 5 , and wherein cytokinin level in said plant is modulated relative to a control plant.  
     
     
         7 . The plant of  claim 6 , wherein said cytokinin level is increased.  
     
     
         8 . The plant of  claim 6 , wherein said cytokinin level is decreased.  
     
     
         9 . The plant of  claim 6 , wherein said polynucleotide is operably linked to a a tissue-preferred promoter, a constitutive promoter, or an inducible promoter.  
     
     
         10 . The plant of  claim 9 , wherein said promoter is a root-preferred promoter, a leaf-preferred promoter, a shoot-preferred promoter, or an inflorescence-preferred promoter.  
     
     
         11 . The plant of  claim 6 , wherein said cytokinin level modulation affects floral development.  
     
     
         12 . The plant of  claim 6 , wherein said cytokinin level modulation affects root development.  
     
     
         13 . The plant of  claim 6 , wherein the plant has an altered shoot-to-root ratio.  
     
     
         14 . The plant of  claim 6 , wherein seed size or seed weight is increased.  
     
     
         15 . The plant of  claim 6 , wherein vigor or biomass yield of said plant is increased.  
     
     
         16 . The plant of  claim 6 , wherein the stress tolerance of said plant is increased.  
     
     
         17 . The plant of  claim 6 , wherein said plant is maize, and tip kernel abortion is reduced.  
     
     
         18 . The plant of  claim 6 , wherein said promoter is stress-insensitive and is expressed in a tissue of the developing seed or related maternal tissue at or about the time of anthesis.  
     
     
         19 . A transformed seed of the plant of  claim 6 .  
     
     
         20 . The plant of  claim 6 , wherein said plant is maize, wheat, rice, barley, sorghum, or rye.  
     
     
         21 . A plant that is genetically modified at a native genomic locus, said genomic locus comprising a polynucleotide of  claim 5 , wherein the cytokinin level of said plant is modulated.  
     
     
         22 . A method of modulating cytokinin level in a plant, comprising transforming said plant with a polynucleotide of  claim 5 , operably linked to a promoter.  
     
     
         23 . The method of  claim 22  wherein said modulation of cytokinin level affects root growth or the shoot-to-root ratio.  
     
     
         24 . The method of  claim 22  wherein said modulation of cytokinin level affects floral development.  
     
     
         25 . The method of  claim 22  wherein said modulation of cytokinin level increases seed size or seed weight.  
     
     
         26 . The method of  claim 22  wherein said modulation of cytokinin level increases plant stress tolerance.  
     
     
         27 . The method of  claim 22  wherein said modulation of cytokinin level affects vigor or biomass yield.  
     
     
         28 . The method of  claim 22  wherein said operably-linked promoter is a tissue-preferred and/or inducible promoter.  
     
     
         29 . The method of  claim 22  wherein said promoter is stress-insensitive and is expressed in a tissue of the developing seed or related maternal tissue at or about the time of anthesis.  
     
     
         30 . The method of  claim 22 , wherein senescence is delayed.  
     
     
         31 . The method of  claim 22  wherein sink strength of the seed of the plant is modulated.  
     
     
         32 . The method of  claim 31  wherein cytokinin level is increased in one or more of the embryo, the endosperm, and tissues proximal thereto.  
     
     
         33 . The method of  claim 32  wherein said proximal tissue comprises the pedicel.  
     
     
         34 . A method for modulating the rate or incidence of shoot regeneration in callus tissue, comprising expressing in said callus tissue a polynucleotide of  claim 5  operably linked to a heterologous promoter.  
     
     
         35 . The method of  claim 33 , wherein said promoter is inducible.  
     
     
         36 . An isolated polynucleotide comprising a nucleotide sequence of SEQ ID NO: 25 or 75 or a functional fragment or variant thereof.  
     
     
         37 . A DNA construct comprising a promoter operably linked to a nucleotide sequence of interest, wherein said promoter comprises the polynucleotide of  claim 36 .  
     
     
         38 . An expression vector comprising the DNA construct of  claim 37 .  
     
     
         39 . A plant comprising at least one DNA construct of  claim 37 .  
     
     
         40 . A method of regulating the expression of a nucleotide sequence of interest, said method comprising introducing into a plant a DNA construct of  claim 37 .  
     
     
         41 . The method of  claim 40  wherein said nucleotide sequence of interest is transcribed to form an RNA molecule which interferes with expression of a homologous native nucleotide sequence.  
     
     
         42 . A method of downregulating expression of ZmIPT1 or ZmIPT2 in a plant, comprising transforming said plant with a construct comprising a promoter operably linked to a polynucleotide which comprises a portion of the polynucleotide of  claim 36 , such that a hairpin molecule is formed which corresponds to the ZmIPT1 promoter or ZmIPT2 promoter.  
     
     
         43 . The method of  claim 42  wherein said promoter is tissue-preferred.