Promoter analysis of the Catharanthus roseus geraniol 10-hydroxylase gene involved in terpenoid indole alkaloid biosynthesis

Abstract

Geraniol 10-hydroxylase (G10H) is an important enzyme in the biosynthetic pathway of monoterpenoid alkaloids found in diverse plant species. The Catharanthus roseus G10H controls the first committed step in biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. The major transcription start site of the promoter was mapped to an adenine 31 bp downstream of the TATA-box. For functional characterization, transcriptional fusions between the G10H promoter fragments with 5′ or 3′ deletions and the GUS reporter gene were generated and their expressions were analyzed in a tobacco protoplast transient expression assay. Deletion of the promoter down to − 318 bp had little effect on GUS activity. However, further deletion of the promoter to position − 103 resulted in approximately 5-fold reduction of GUS activity. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between − 191 and − 147, − 266 and − 188, and − 318 and − 266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings GUS expression was tissue-specific, restricted to leaf and actively growing cells around the root tip, and not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitor and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade.