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

Abstract

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 A1510 to A8, 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 A632 had little eect on gusA expression. However, deletion to position A366 resulted in a complete loss of basal activity and largely eliminated elicitor-induced expression, indicating that the region from A632 to A366 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 (A632 to A366), is suggestive of a functional role for this factor in basal expression and elicitor responsiveness of the Cpr promoter.

Nuclear factors GT-1 and 3AF1 interact with multiple sequences within the promoter of the Tdc gene from Madagascar periwinkle: GT-1 is involved in UV light-induced expression

“…GT-1 was also shown to interact with the promoter of the pathogenesis-related gene PR1a (Buchel et al 1996). Furthermore, GT-1 interacts with two other genes, Str and Cpr, that operate in the C. roseus TIA pathway (Lopes-Cardoso et al 1997;Pasquali et al 1999). Thus, the available binding studies suggest that GT-1 represents a regulator not only of light-regulated, but also of defence-related genes.…”

“…Our results show that the previously described factors GT-1 (Green et al 1987) and 3AF1 bind to several Tdc promoter fragments. Interestingly, GT-1 was also shown to interact with important enhancer regions in the Str (Pasquali et al 1999) and Cpr (Lopes-Cardoso et al 1997) genes. To investigate the role of GT-1 in regulating the activity of the Tdc promoter in vivo, we employed a strategy in which multiple binding sites in an important enhancer were mutagenized.…”

“…At present, cDNA and genomic clones for the TIA biosynthetic genes Tdc (De Luca et al 1989;Goddijn et al 1994), Str (McKnight et al 1990;Pasquali et al 1992Pasquali et al , 1999 and Cpr (Meijer et al 1993b;Lopes-Cardoso et al 1997) have been isolated from C. roseus. In cell suspensions, the Tdc, Str and Cpr genes are coordinately induced by fungal elicitors (Pasquali et al 1992;Roewer et al 1992;Meijer et al 1993b), and repressed by auxin Pasquali et al 1992;Meijer 1993).…”

Testifying the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAγ1

“…Interestingly, the Box II-related sequences may have either a positive or negative role in the transcription of different genes in different organs. For example, GT-1 was shown to function as a silencer to interact with the Box II element of bean's CHS promoter, and the GT-1 binding activity with the tobacco PR-1a promoter was reduced after challenging with salicylic acid or tobacco mosaic virus (Dron et al 1988;Lawton et al 1991;Villain et al 1994Villain et al , 1996Buchel et al 1996;Lopes Cardoso et al 1997). On the other side, it is also known that the R-gene products can interact with pathogen elicitors either directly or indirectly (Tang et al 1996;Baker et al 1997;Jia et al 2000;Jones 2001;Nimchuk et al 2001).…”

Molecular cloning and structural analysis of the cucumber (Cucumis sativus L.) phosphoenolpyruvate carboxykinase (CsPCK) gene

“…Light activation and calcium/calmodulin stimulation can result in two GT-1 binding boxes (5'-C;GTI-AA) being sandwiched between two pyrimidine boxes, from -337 bp on the coding strand and from -368 bp on the complementary strand (Lam and Chua, 1990;Gourrierec et al, 1999;Zhou, 1999). These GT-1 core elements are possibly involved in the expression of both CsPCK and ms during cucumber development, as shown by the transcription of multiple genes in various organs (Eyal et al, 1995;Buchel et al, 1996, Lopes Cardoso et al, 1997. Together with a I-box, eight GT-1 boxes were found in thaumatin-like gene (MdTL1) promoter in apple (Kim et al, 2003).…”