Two Nicotiana plumbaginifolia cDNA clones, CYP72A2 and npl2, with high sequence similarity to cytochrome P450 monooxygenases were isolated using reverse transcription-polymerase chain reaction. CYP72A2 has an open reading frame of 1524 nucleotides and its deduced 508 amino acid sequence has 45% identity to Catharanthus roseus P450 CYP72A1. npl2 is similar to CYP72A2, except for an 82-nucleotide deletion within its coding region and an internal stop codon. Southern blot analysis indicated that there are at least 3 copies of the CYP72A2 gene and that they are induced at the RNA and protein levels by mechanical wounding, insect chewing (Manduca sexta) and cytokinin application. The response to feeding larvae and wounding was systemic. In N. plumbaginifolia plants transformed with a wound-inducible cytokinin biosynthesis gene construct (PI-II-ipt), gene induction in response to wounding and feeding insects occurred sooner and was generally higher than in untransformed plants. The modulation of CYP72A2 expression by cytokinins and the possible role of P450 in plant defense responses are discussed.
