Identification of UV-B light-responsive regions in the promoter of the tryptophan decarboxylase gene from Catharanthus roseus.

The tryptophan decarboxylase (Tdc) gene encodes a key enzyme in the biosynthesis of terpenoid indole alkaloids (TIAs) in Catharanthus roseus. TIAs absorb ultraviolet light (UV) and putative functions in plants include a role as UV protectants. In support of this possible function we demonstrate here that UV light induces accumulation of several TIAs as well as expression of the Tdc gene in C. roseus. In addition, in tobacco a Tdc-gusA construct was found to be specifically induced by UV-B light. Lack of induction by UV-A or other wavelengths of light indicate that Tdc expression is regulated by a specific UV-B receptor and corresponding signal transduction pathway. To identify UV-responsive Tdc promoter elements, a loss-of-function analysis was performed, in which deletion derivatives were fused to the gusA reporter gene and analysed in transgenic tobacco plants. Truncation of the Tdc promoter from -1818 (relative to the start of transcription) to -160 reduced expression levels two-fold without affecting the qualitative UV response. Deletion to -37 further reduced expression levels five-fold, but the delta37 promoter also remained UV-responsive. Subsequently, the -160 to -37 region was further studied by gain-of-function experiments, in which the transcriptional activities of tetramerized subfragments fused to truncated promoters were analysed. Combination of the data identified several functional regions in the -160 to +198 promoter. The - 160 to -99 region acts as the main transcriptional enhancer. UV-responsive elements appeared to be redundant in the -160 Tdc promoter and to reside between -99 and -37 and between -37 and + 198.

Alkaloid production by a Cinchona officinalis 'Ledgeriana' hairy root culture containing constitutive expression constructs of tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus.

Cinchona officinalis 'Ledgeriana', former called Cinchona ledgeriana, hairy roots were initiated containing constitutive-expression constructs of cDNAs encoding the enzymes tryptophan decarboxylase (TDC) and strictosidine synthase (STR) from Catharanthus roseus, two key enzymes in terpenoid indole and quinoline alkaloid biosynthesis. The successful integration of these genes and the reporter gene gus-int was demonstrated using Southern blotting and the polymerase chain reaction. The products of TDC and STR, tryptamine and strictosidine, were found in high amounts, 1200 and 1950 µg g-1 dry weight, respectively. Quinine and quinidine levels were found to rise up to 500 and 1000 µg g-1 dry weight, respectively. The results show that genetic engineering with multiple genes is well possible in hairy roots of C. officinalis. However, 1 year after analyzing the hairy roots for the first time, they had completely lost their capacity to accumulate alkaloids.

Expression of two consecutive genes of a secondary metabolic pathway in transgenic tobacco: molecular diversity influences levels of expression and product accumulation.

We have created a population of transgenic tobacco plants carrying cDNAs encoding two consecutive enzymes from early stages in monoterpenoid alkaloid biosynthesis in Catharanthus roseus. The cDNAs, encoding tryptophan decarboxylase (tdc) and strictosidine synthase (str1) together with a selectable marker gene, were introduced on a single transforming plasmid into tobacco leaves by particle bombardment. Analysis of 150 independent transgenic plants at the DNA and RNA levels demonstrated a range of integration events and steady-state transcript levels for the tdc and str1 transgenes. Southern blot analysis indicated that the tdc and str1 transgenes were integrated at least once in all 150 transformants giving a 100% co-integration frequency of the two unselected genes carried on the same plasmid. A comparison of Southern and northern data suggested that in 26% of the plants, both tdc and str1 transgenes were silenced, 41% demonstrated a preferential silencing of either the tdc or the str1 transgene, with the remaining 33% of the plants expressing both transgenes. We observed no clear correlation between the number of integration events of a specific transgene and the levels of accumulated transcript. Twenty plants representing the range of molecular diversity in the transgenic population were selected for further analysis. Seeds were collected from self-fertilised transformants and germinated on medium containing kanamycin. Seedlings were harvested after 7 weeks and TDC and STR1 enzymatic assays were carried out. We observed a 24- and 110-fold variation in levels of TDC and STR1 activities, respectively. Our data correlate molecular diversity with biochemistry and accumulation of end-product and provide a detailed molecular and biochemical characterization of transgenic plants transformed with a single plasmid carrying two genes of secondary metabolism.

Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.

Suspension cultured transgenic cells of Nicotiana tabacum expressing tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus produce strictosidine upon secologanin feeding.

A transgenic cell suspension culture of Nicotiana tabacum L. `Petit Havana' SR1 was established expressing tryptophan decarboxylase and strictosidine synthase cDNA clones from Catharanthus roseus (L.) G. Don under the direction of cauliflower mosaic virus 35S promoter and nopaline synthase terminator sequences. During a growth cycle, the transgenic tobacco cells showed relatively constant tryptophan decarboxylase activity and an about two- to sixfold higher strictosidine synthase activity, enzyme activities not detectable in untransformed tobacco cells. The transgenic culture accumulated tryptamine and produced strictosidine upon feeding of secologanin, demonstrating the in vivo functionality of the two transgene-encoded enzymes. The accumulation of strictosidine, which occurred predominantly in the medium, could be enhanced by feeding both secologanin and tryptamine. No strictosidine synthase activity was detected in the medium, indicating the involvement of secologanin uptake and strictosidine release by the cells.