Characterization of a polyclonal antiserum against the monoterpene monooxygenase, geraniol 10-hydroxylase from Catharanthus roseus

Geraniol 10-hydroxylase (G10H) is a P450 containing enzyme which is the first committed step in the biosynthesis of monoterpene indole alkaloids (MIAs), including the Catharanthus roseus-anticancer drugs vinblastine and vincristine. It is thought that G10H has a regulatory role in MIA production. In the present paper, we report the characterization of a polyclonal serum raised against the purified G10H polypeptide. Anti-G10H IgG was able to inhibit the G10H activity and also recognized the G10H polypeptide from C. roseus and other plants producing MIAs. These results establish the usefulness of this antiserum as a biochemical tool for the study of G10H regulation.     

Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis.

Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti-cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.     

Identification of a low vindoline accumulating cultivar of Catharanthus roseus (L.) G. Don by alkaloid and enzymatic profiling

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is the only source of the monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, key pharmaceutical compounds used to combat a number of different cancers. The present study uses high performance liquid chromatography for metabolic profiling of the MIAs extracted from seedlings and young leaves of 50 different flowering cultivars of C. roseus to show that, except for a single low vindoline cultivar (Vinca Mediterranean DP Orchid), they accumulate similar levels of MIAs. Further enzymatic studies with extracts from young leaves and from developing seedlings show that the low vindoline cultivar has a 10-fold lower tabersonine-16-hydroxylase activity than those of C. roseus cv. Little Delicata. It is concluded that rapid metabolic and more selective enzymatic profiling of Catharanthus mutants could be useful for the identification of a range of altered MIA biosynthesis lines.     

Molecular cloning and characterisation of two calmodulin isoforms of the Madagascar periwinkle Catharanthus roseus

Involvement of Ca(2+) signalling in regulation of the biosynthesis of monoterpene indole alkaloids (MIA) in Catharanthus roseus has been extensively studied in recent years, albeit no protein of this signalling pathway has been isolated. Using a PCR strategy, two C. roseus cDNAs encoding distinct calmodulin (CAM) isoforms were cloned and named CAM1 and CAM2. The deduced 149 amino acid sequences possess four Ca(2+) binding domains and exhibit a close identity with Arabidopsis CAM isoforms (>91%). The ability of CAM1 and CAM2 to bind Ca(2+) was demonstrated following expression of the corresponding recombinant proteins. Furthermore, transient expression of CAM1-GFP and CAM2-GFP in C. roseus cells showed a typical nucleo-cytoplasm localisation of both CAMs, in agreement with the wide distribution of CAM target proteins. Using RNA blot analysis, we showed that CAM1 and CAM2 genes had a broad pattern of expression in C. roseus organs and are constitutively expressed during a C. roseus cell culture cycle, with a slight inhibitory effect of auxin for CAM1. Using RNA in situ hybridisation, we also detected CAM1 and CAM2 mRNA in the vascular bundle region of young seedling cotyledons. Finally, using specific inhibitors, we also showed that CAMs are required for MIA biosynthesis in C. roseus cells by acting on regulation of expression of genes encoding enzymes that catalyse early steps of MIA biosynthesis, such as 1-deoxy-d-xylulose 5-phosphate reductoisomerase and geraniol 10-hydroxylase.     

Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots

In plants, the indole pathway provides precursors for a variety of secondary metabolites. In Catharanthus roseus, a decarboxylated derivative of tryptophan, tryptamine, is a building block for the biosynthesis of terpenoid indole alkaloids. Previously, we manipulated the indole pathway by introducing an Arabidopsis feedback-insensitive anthranilate synthase (AS) alpha subunit (trp5) cDNA and C. roseus tryptophan decarboxylase gene (TDC) under the control of a glucocorticoid-inducible promoter into C. roseus hairy roots [Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004a. Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels. Biotechnol. Bioeng. 86, 718-727; Hughes, E.H., Hong, S.-B., Gibson, S.I., Shanks, J.V., San, K.-Y. 2004b. Metabolic engineering of the indole pathway in Catharanthus roseus hairy roots and increased accumulation of tryptamine and serpentine. Metabol. Eng. 6, 268-276]. Inducible expression of either or both transgenes did not lead to significant increases in overall alkaloid levels despite the considerable accumulation of tryptophan and tryptamine. In an attempt to more successfully engineer the indole pathway, a wild type Arabidopsis ASbeta subunit (ASB1) cDNA was constitutively expressed along with the inducible expression of trp5 and TDC in C. roseus hairy roots. Transgenic hairy roots expressing both trp5 and ASB1 show a significantly greater resistance to feedback inhibition of AS activity by tryptophan than plants expressing only trp5. In fact, a 4.5-fold higher concentration of tryptophan is required to achieve 50% inhibition of AS activity in plants overexpressing both genes than in plants expressing only trp5. In addition, upon a 3 day induction during the exponential phase, a trp5:ASB1 hairy root line produced 1.8 times more tryptophan (specific yield ca. 3.0 mg g(-1) dry weight) than the trp5 hairy root line. Concurrently, tryptamine levels increase up to 9-fold in the induced trp5:ASB1 line (specific yield ca. 1.9 mg g(-1) dry weight) as compared with only a 4-fold tryptamine increase in the induced trp5 line (specific yield ca. 0.3 mg g(-1) dry weight). However, endogenous TDC activities of both trp5:ASB1 and trp5 lines remain unchanged irrespective of induction. When TDC is ectopically expressed together with trp5 and ASB1, the induced trp5:ASB1:TDC hairy root line accumulates tryptamine up to 14-fold higher than the uninduced line. In parallel with the remarkable accumulation of tryptamine upon induction, alkaloid accumulation levels were significantly changed depending on the duration and dosage of induction.