Modulation of cellular polyamines in tobacco by transfer and expression of mouse ornithine decarboxylase cDNA.
In an attempt to modulate the metabolism of polyamines in plants, Agrobacterium tumefaciens strains were produced which contained either a full-length or a 3'-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of the cauliflower mosaic virus 35S promoter. Plants of Nicotiana tabacum cv. Xanthi were used for transformation with these two strains of Agrobacterium. Transformations were confirmed by Southern hybridization and amplification by polymerase chain reaction. Two plants containing the full-length cDNA (ODC-12 and ODC-30) and two containing the truncated cDNA (12701-2 and 12701-31) were selected for further experiments. Northern blot analysis indicated that transcription was occurring and western blot analysis detected a polypeptide of ca. 50 kDa that was unique to the plants transformed with truncated ODC cDNA. In order to distinguish between the native and the mouse ODC in the transformed tissues, enzyme activity was assayed at pH optima for the two enzymes, i.e. pH 8.2 and 6.8, respectively. Substantially higher levels of ODC activity were seen at pH 6.8 (optimum for mouse ODC) in the transformants as compared to the controls. This ODC activity was inhibited by alpha-difluoromethylornithine and anti-mouse ODC antisera in a manner consistent with that reported for the mouse ODC. Analysis of cellular polyamines showed significantly elevated levels (4-12-fold) of putrescine in callus derived from transformed plant tissues as compared to the controls. The modulation of polyamine biosynthesis in plants by these techniques should allow us to further analyze the role of these ubiquitous compounds in plant growth and development.
DeScenzo
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RA
Department of Plant Biology, University of New Hampshire, Durham 03824.
Minocha
S C
SC
D016960:Agrobacterium tumefaciens / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D051379:Mice; D008969:Molecular Sequence Data; D009955:Ornithine Decarboxylase / Q000096:biosynthesis* / Q000235:genetics; D065108:Ornithine Decarboxylase Inhibitors; D010446:Peptide Fragments / Q000096:biosynthesis; D030821:Plants, Genetically Modified / Q000378:metabolism*; D010947:Plants, Toxic; D011073:Polyamines / Q000032:analysis*; D011700:Putrescine / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D065108:Ornithine Decarboxylase Inhibitors; D010446:Peptide Fragments; D011073:Polyamines; D011994:Recombinant Proteins; D009955:Ornithine Decarboxylase; D011700:Putrescine