Medium-Induced Formation of Indole Alkaloids and Concomitant Changes of Interrelated Enzyme Activities in Cell Suspension Cultures of Catharanthus roseus

124

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 ...

Section: Discussionsupporting

confidence: 92%

Developmental Regulation of Enzymes of Indole Alkaloid Biosynthesis in Catharanthus roseus

Luca¹,

Fernández²,

Campbell³

et al. 1988

124

“…The enzyme TDC3 (EC 4.1.1.28) converts Trp to tryptamine in plants (13). Tryptamine provides the indole unit of monoterpenoid-indole and derived alkaloids.…”

mentioning

confidence: 99%

“…This peak in TDC activity is followed by an almost immediate accumulation of alkaloid end products (1, 5). TDC activity is also highly regulated in Catharanthus and Cinchona cell culture, where under certain conditions, transient activation of the enzyme can occur (8,13,16,21). The appearance of this enzyme in Catharanthus cultures has been shown to be preceded by transient appearance of TDC mRNA after induction by fungal elicitors (20).…”

mentioning

confidence: 99%

Auxins Induce Tryptophan Decarboxylase Activity in Radicles of Catharanthus Seedlings

Aerts

Alarco²,

Luca³

1992

Germinating seedlings of Catharanthus roseus produce monoterpenoid indole alkaloids as a result of a transient increase of tryptophan decarboxylase (TDC) activity. The influence of auxins on this transient rise of TDC activity was studied. External application of indolebutyric acid or 2,4-dichlorophenoxyacetic acid at a concentration of 20 to 40 Mm enhanced and prolonged the rise in TDC activity in developing seedlings. Auxin treatment also influenced the morphology of the seedlings; it induced a shortening and thickening of the hypocotyl and the radicle and promoted the initiation of lateral roots in the radicle. During development, the radicles of auxin-treated seedlings displayed a gradual increase in TDC activity that was absent in the radicles of untreated controls. Examination of immunoblots revealed anti-TDC reactive proteins in extracts from radicles of auxin-treated seedlings, but none in extracts from radicles of control seedlings. In contrast, TDC activity and immunoreactive protein levels in the aerial parts of controls and auxin-treated seedlings were comparable. Our results indicate that externally applied auxins induce both abnormal development and TDC activity in the radicles of Catharanthus seedlings. Although auxins slightly delayed the light-mediated induction of the cotyledon-specific last step in vindoline biosynthesis (i.e. acetylcoenzyme A: deacetylvindolin-O-acetyltransferase activity), seedlings still synthesized vindoline, one of the major alkaloid end products.

“…The enzyme has previously been characterized and purified to homogeneity from cell suspension cultures of Catharanthus roseus (18) indole alkaloids ajmalicine and catharanthine. It has been speculated that expression of TDC is the controlling factor for alkaloid production in Catharanthus cell suspension cultures (14,19). However, recent studies suggest that although expression of TDC is highly regulated in plant tissue cultures (5,8,9,14,17), as in intact plants (4,6), appearance of TDC enzyme activity in culture does not always result in the accumulation of indole alkaloids (9,15).…”

mentioning

confidence: 99%

Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings

Fernández

Owen²,

Kurz³

et al. 1989

L-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Westem immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program.