A PROMOTER REGION THAT CONTROLS BASAL AND ELICITOR-INDUCIBLE EXPRESSION LEVELS OF THE NADPH-CYTOCHROME P450 REDUCTASE GENE (CPR) FROM CATHARANTHUS-ROSEUS BINDS NUCLEAR FACTOR GT-1

NADPH:cytochrome P450 reductase (CPR) is essential for the activation of cytochrome P450 enzymes which are involved in a wide variety of met abolic pathways in plants. including those related to defence response s. In the subtropical plant Catharanthus roseus several cytochrome P45 0 enzymes operate in the biosynthesis of defence-related terpenoid ind ole alkaloids (TIAs). In agreement with the importance of CPR in defen ce, Cpr mRNA levels in C. roseus were found to be enhanced by fungal e licitor preparations that also induce TIA biosynthesis and P450 gene e xpression. Here we describe the isolation of a C. roseus genomic DNA c lone covering the 5' part of the Cpr gene and 1.6-kb of upstream seque nces. Mapping of the transcription start site showed the untranslated leader sequence is approximately 280 bp long. To study the control of gene expression by the Cpr promoter, transcriptional fusions between C pr promoter fragments and the gusA reporter gene were generated and th eir expression was analyzed in stably transformed tobacco planes. The Cpr promoter fragment extending fi-om -1510 to -8, with respect to the ATG start codon, conferred basal and elicitor-inducible expression on the gusA reporter gene, strongly indicating that the Cpr gene of C. r oseus is indeed controlled by this promoter region. Progressive deleti on from the 5' end of the promoter to position -632 had little effect on gusA expression. However, deletion to position -366 resulted in a c omplete loss of basal activity and largely eliminated elicitor-induced expression, indicating that the region from -632 to -366 contains the main transcription-enhancing cis-regulatory sequences. Electrophoreti c mobility shift assays with tobacco nuclear extracts showed that bind ing sites for nuclear factor GT-1 are redundant in the Cpi promoter, b ut absent from the downstream part of the leader sequence. The presenc e of strong GT-1 binding sites in the main enhancer region (-632 to -3 66), is suggestive of a functional role for this factor in basal expre ssion and elicitor responsiveness of the Cpr promoter.