Developing seedlings of Catharanthus roseus were analyzed for appearance of tryptophan decarboxylase (TDC), strictosidine synthase (SS), N-methyltransferase (NMT) and O-acetyltransferase (DAT) enzyme activities. SS enzyme activity appeared early after germination and was present throughout most of the developmental study. TDC activity was highly regulated and peaked over a 48 hour period achieving a maximum by day of 5 of seedling development. Both TDC and SS were present in all tissues of the seedling. NMT and DAT enzyme activities were induced after TDC and SS had peaked and these activities could only be found in hypocotyls and cotyledons. TDC, SS, and NMT did not require light for induction whereas DAT enzyme activity was increased approximately 10-fold after light treatment of dark grown seedlings.The expression of indole alkaloid biosynthetic capability in Catharanthus roseus (L.) G. Don cv Little Delicata has recently been shown to be under developmental control in germinating seedlings (1-3). These studies have demonstrated that while the majority of genes controlling indole alkaloid biosynthesis were expressed early in germination and in all plant tissues, those involved in the late stages of vindoline biosynthesis were only expressed in the aerial parts of the plant, might be expressed later in development than the rest of the pathway, and might be under light regulation.The tissue-specific localization of the late stages of vindoline biosynthesis in the intact plant was further elucidated when it was discovered that the NMT4 which catalyzes the third last step in vindoline biosynthesis is possibly localized in the thylakoids of chloroplasts (4) (Fig. 1). DAT, which is responsible for transforming deacetylvindoline to vindoline, the last step in the vindoline pathway is, however, localized in the cytosol (4). TDC and SS, two of the key enzymes involved in the early stages of indole alkaloid biosynthesis, are also cytosolic (4) (Fig. 1). It is not known where the other steps in vindoline biosynthesis occur within the cell (Fig. 1).The present investigation extends the previous studies by Enzyme Extraction. Fifty seedlings or parts thereof were thawed in 1.25 ml grinding buffer containing 100 mM Hepes (pH 7.6), 1 mm DTT, and 5 mM EDTA. The plant material was homogenized for 30 to 40 s with an Ultra-Turrax drive equipped with a 1 to 10 ml shaft. After homogenization, the slurry was transferred to 1.5 ml microfuge tubes and centrifuged for 3 min in an Eppendorf model 5412 microcentrifuge. The clear supernatant was transferred to clean tubes and 1 ml aliquots were applied to Pharmacia PD-10 columns preequilibrated with grinding buffer lacking EDTA. Two ml desalted samples were collected ensuring elimination of internal substrates present in the crude extracts.Enzyme Assays. DAT was assayed as described previously (2).The assay mixture consisted of 5 ,uM deacetylvindoline, 4.4 jM [1-'4C]acetyl coenzyme A (0.045 gCi) and enzyme in a total of 100 gl 0.1 M Tris-HCI (pH 8). The mixture was incubated for ...

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Catharanthus roseus hairy root cultures, genetically transformed with Agrobacterium rhizogenes, produce a wide variety of indole alkaloids. The effect of sucrose, phosphate, nitrate, and ammonia concentrations on growth and indole alkaloid production of C. roseus hairy root cultures were studied by using statistical experimental designs and linear regression analysis. Contradictory effects of these nutrients on growth and indole alkaloid production were found. The maximal growth was obtained by having 77.8 mg NaH2P04 * H 2 0 / L and 1.311 g K N 0 3 / L in the medium, whereas the specific production of alkaloids was highest at the lowest levels of all the nutrients studied. The maximal dry weight was obtained with high values of sucrose and ammonia, but clear optimum concentrations could not be found. When having enough nutrients to support reasonable growth, it appeared difficult to affect the specific alkaloid production rates considerably. The growth (dry wt.) with the optimized nutrient concentrations in the medium was more than 50% better than in the control medium with about the same alkaloid production.