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Submitted to: Plant Molecular Biology International Conference Proceedings
Publication Type: Abstract Only Publication Acceptance Date: 10/27/2000 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: A Nicotiana plumbaginifolia cDNA clone, CYP72A2, with high sequence similarity to cytochrome P450 monooxygenases was isolated using reverse transcription-polymerase chain reaction. CYP72A2 has an open reading frame of 1524 nucleotides and belongs to a small gene family. Its deduced 508 amino acid sequence shares 45% identity with the Catharanthus roseus cytochrome P450 CYP72A1 that has been tentatively assigned as geraniol-10-hydroxylase, an enzyme that catalyzes the conversion of geraniol to 10-hydroxygeraniol, the rate limiting step in the biosynthesis of the monoterpene alkaloids camptothecin, vinblastine and vincristine. Mechanical wounding, insect chewing (Manduca sexta) and cytokinin application were shown to induce CYP72A2 expression. A higher level and more rapid induction of CYP72A2 transcripts was observed in N. plumbaginifolia plants transformed with a wound-inducible cytokinin biosynthesis gene construct (PI-II-ipt) as compared to controls. Mechanical wounding of the PI-II-ipt leaves induced a 6-fold increase of CYP72A2 messages at 6 h in comparison to only a 2-fold induction observed after 12 h in untransformed plants. A similar response was observed when plants were sprayed with either 5 x 10-6 M zeatin or when M. sexta larvae fed on the leaves. The up-regulation of the CYP72A2 transcripts in response to insect or mechanical wounding was systemic. Polyclonal antibodies specific for three internal regions of the deduced CYP72A2 protein cross-reacted with a 58.8 kDa polypeptide that accumulated in response to wounding. Functional studies are in progress to determine if CYP72A2 has geraniol-10-hydroxylase activity. |
