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Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Abstract Only Publication Acceptance Date: 6/27/1998 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Cytochrome P450 monooxygenases are a large group of heme-containing enzymes that mediate a wide range of oxidative reactions involved in the biosynthetic catabolic and detoxification pathways of all living organisms. Plant P450s catalyze the synthesis of a variety of secondary products, some of which are shown to inhibit insects, pathogens and animal herbivores. Using RT-PCR of total RNA from Nicotiana plumbaginifolia transformed with the isopentenyl transferase (ipt) gene that is fused to a wound-inducible promoter, two full length clones of P450 were isolated and sequenced. One of the clones is bigger than the other by 81 nucleotides and its predicted 508 amino acid sequence has 44% identity to Catharanthus roseus P450 (CYP72), a protein exhibiting geraniol 10-hydroxylase activity. When in vitro transcribed and translated using the TNT wheat germ extract system, two 35S-met or 3H-leu labeled polypeptides with molecular masses of 53 and 34 kDa were obtained using clones and 1 and 2, respectively. The expressio of P450 is regulated by light and darkness. Northern blot analysis revealed that transcript accumulation was maximum during mid-day but was lowest at night. Hornworm (Manduca sexta) feeding and mechanical wounding disrupted this rhythm resulting in an elevated level of expression at night in the wounded leaf. The level of induction was 4-to 6- fold higher in ipt-transformed leaves after 6-12 hr of mechanical wounding in comparison to 2- to 3.5 fold induction from wounded but untransformed leaves. The response to insect feeding and mechanical wounding was systemic and was detected maximally in the leaf immediately above the damaged leaf. The role of P450 in plant defense responses will be explored by transforming plants with various P450 sense and antisense constructs. |
