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 metabolic
pathways in plants, including those related to defence
responses. In the subtropical plant Catharanthus roseus
several cytochrome P450 enzymes operate in the biosynthesis of defence-related terpenoid indole alkaloids
(TIAs). In agreement with the importance of CPR in
defence, Cpr mRNA levels in C. roseus were found to be
enhanced by fungal elicitor preparations that also induce
TIA biosynthesis and P450 gene expression. Here we
describe the isolation of a C. roseus genomic DNA clone
covering the 5¢ part of the Cpr gene and 1.6-kb of upstream sequences. 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 Cpr promoter fragments and the gusA reporter
gene were generated and their expression was analyzed
in stably transformed tobacco plants. The Cpr promoter
fragment extending from )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. roseus is indeed controlled by this promoter region. Progressive deletion
from the 5¢ end of the promoter to position )632 had little e ect on gusA expression. However, deletion to
position )366 resulted in a complete 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. Electrophoretic mobility shift assays with tobacco nuclear extracts showed that binding sites for
nuclear factor GT-1 are redundant in the Cpr promoter,
but absent from the downstream part of the leader sequence. The presence of strong GT-1 binding sites in the
main enhancer region ()632 to )366), is suggestive of a
functional role for this factor in basal expression and
elicitor responsiveness of the Cpr promoter.info:eu-repo/semantics/publishedVersio