Subcellular Localization of Enzymes Involved in Indole Alkaloid Biosynthesis in Catharanthus roseus

The subcellular localization of enzymes involved in indole alkaloid biosynthesis in leaves of Catharanthus roseus has been investigated. Tryptophan decarboxylase and strictosidine synthase which together produce strictosidine, the first indole alkaloid of this pathway, are both cytoplasmic enzymes. S-Adenosyl-l-methionine: 16-methoxy-2,3-dihydro-3-hydroxytabersonine-N-methyltransferase which catalyses the third to last step in vindoline biosynthesis could be localized in the chloroplasts of Catharanthus leaves and is specifically associated with thylakoids. Acetyl-coenzyme-A-deacetylvindoline-O-acetyltransferase which catalyses the last step in vindoline biosynthesis could also be localized in the cytoplasm. The participation of the chloroplast in this pathway suggests that indole alkaloid intermediates enter and exit this compartment during the biosynthesis of vindoline.     

Some alkaloids of Alstonia undulata

Sixteen indole alkaloids have been identified in the root bark and in the leaves of Alstonia undulata from New Caledonia. They are vincamedine, tetrahydroalstonine, alstonidine, deplancheine, fluorocarpamine, pleiocarpamine, 11-methoxyakuammicine, cabucraline, desoxycabufiline, cabucraline-N(4)-oxide, 11-methoxyakuammicine N(4)-oxide, gentiacraline, plumocraline, vincorine, cathafoline and pericyclivine. Five bis indoles of unknown structure have been isolated. The root bark alkaloid mixture contains gentiacraline the first alkaloid made up of an indole and a pyridine alkaloid.     

Lochvinerine: A new indole alkaloid of Vinca major

Five indole alkaloids have been isolated from the aerial parts of Vinca major. Four of these have been identified as reserpinine (pubescine), vincamajoreine, majoridine and 10-methoxyvellosimine. The fifth base, a new indole alkaloid lochvinerine, has been assigned the depicted structure.     

A chemotaxonomic division of Murraya based on the distribution of the alkaloids yuehchukene and girinimbine

An examination of the roots of eight species and one variety of Murraya for the presence of the dimeric indole yuehchukene has revealed a dichotomy in the genus between species producing this alkaloid and those producing the carbazole alkaloid girinimbine, with no apparent overlap between the two groups of species.     

Ervatinine,an indole alkaloid from Ervatamia coronaria

An indole alkaloid, ervatinine, has been isolated from the leaves of Ervatamia coronaria and its structure has been elucidated.     

Stapfinine,an indole alkaloid from Ervatamia coronaria

A new indole alkaloid, stapfinine, was isolated from the leaves of Ervatamia coronaria and its structure determined by spectroscopic means.     

3-dehydromitragynine: An alkaloid from Mitragyna speciosa

An investigation of the fresh leaves of Mitragyna speciosa has resulted in the isolation of a new alkaloid in addition to the indole alkaloids previously reported. The new alkaloid is the 3-dehydro derivative of mitragynine and its structure was elucidated by spectral means and chemical transformations. (-)-Epicatechin was also isolated from the leaves.     

Contribution of indole alkaloids to Sitobion avenae (F.) resistance in wheat

Abstract:  Indole alkaloid contents of five winter wheat varieties at different growth stages, and the relationship between indole alkaloids and carboxylesterase activities of Sitobion avenae (F.) from wheat ears of different varieties were studied. The results indicated that KOK1679 and My295, highly and moderately aphid-resistant varieties, had a high indole alkaloid content during vegetative growth. During their reproduction growth, resistant varieties had a high alkaloid content in penultimate leaves, and indole alkaloid contents of KOK1679 were significantly higher than that of other varieties in wheat ears. The indole alkaloid contents of all varieties were low in flag leaves except for Han4564 . A significant correlation was shown between carboxylesterase activity in aphids collected from wheat ears and indole alkaloid contents in ears of the wheat varieties ( r  = 0.9646).     

The development of a single-stage growth and indole alkaloid production medium for Catharanthus roseus (L.) G. Don suspension cultures

By modification of a standard Murashige and Skoog plant tissue culture maintenance medium, a system has been developed for Catharanthus roseus cell suspension cultures in which both growth and indole alkaloid accumulation can occur in a single-stage culture of 14–21 days. Precise optimization of the medium depends upon the cell line under investigation, but the inclusion of lactose as the carbohydrate source, and NAA and kinetin as growth regulators, will generally increase yields of the indole alkaloid catharanthine. Treatment of cells growing in this optimized medium with agents that stimulate the accumulation of secondary metabolites both increases the yield of catharanthine and reduces the time required for production. We believe that this process could be useful for the commercial production of plant secondary metabolites.     

Effects of bioregulators on indole alkaloid biosynthesis in Catharanthus roseus cell culture

Several carotenoid-inducers are effective in promoting indole alkaloid formation in Catharanthtus roseus cell culture. Among the five compounds tested, viz. 1,1-dimethylpiperidine, 2-diethylaminoethyl-3,4-dichlorophenylether, 2-diethylaminoethyl-2,4-dichlorophenylether, 2-diethylaminoethyl-β-naphthylether and 2-diethylaminoethyl-3,4-dimethylphenylether, 1,1-dimethylpiperidine, 2-diethylaminoethyl-2,4-dichlorophenylether and 2-diethylaminoethyl-β-naphthylether at 5 ppm concentration increased total alkaloid production by up to ca 20 % with concomitant increases in ajmalicine and catharanthine. Concentrations of 2-diethylaminoethyl-2,4-dichlorophenylether higher than 5 ppm caused growth inhibition and decrease in alkaloid synthesis.     

A new alkaloid from Voacanga chalotiana

The structure of 3-hydroxyvoachalotine, a new indole alkaloid isolated from the root bark of Voacanga chalotiana, has been determined by spectroscopic analysis and by chemical correlation with voachalotine.     

Indole alkaloids from Rauvolfia media

Four monomeric indole alkaloids have been isolated from the bark of Rauvolfia media. Three of them are the known cabucine, reserpiline and mauiensine; the fourth is a new alkaloid, 12-hydroxymauiensine.     

Isolation and Characterization of a 2-Oxoglutarate Dependent Dioxygenase Involved in the Second-to-Last Step in Vindoline Biosynthesis

Young leaves from Catharanthus roseus plants contain the enzymes which convert the monoterpenoid indole alkaloid, tabersonine by three hydroxylations, two methylations, and one acetylation step to vindoline. A novel direct enzyme assay has been developed for a hydroxylase involved in vindoline biosynthesis, which catalyzes the C4-hydroxylation of 2,3-dihydro-3-hydroxy-N(1)-methyltabersonine to the 3,4-dihydroxy derivative. The enzyme showed an absolute requirement for 2-oxoglutarate and enzymatic activity was enhanced by ascorbate, establishing it as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). The hydroxylase exhibited specificity for position 4 of various alkaloid substrates. The enzyme exhibited a pH optima between 7 and 8 and an apparent molecular weight of 45,000. The appearance of 4-hydroxylase activity was developmentally regulated and was shown to be inducible by light treatment of seedlings. Substrate specificity studies of this enzyme for indole alkaloid substrate suggested that hydroxylation at position 3 and N-methylation occur prior to hydroxylation at position 4. This is in agreement with previous studies which suggest that C4-hydroxylation is the second to last step in vindoline biosynthesis in Catharanthus roseus.     

Rhazimanine,an indole alkaloid from fruits of Rhazya stricta

A new indole alkaloid, rhazimanine, has been isolated from the fruits of Rhazya stricta. It belongs to the Corynanthe group of alkaloids with a cis-quinolizidine system bearing an H-3 in the β-configuration, equatorial to ring ‘D’. The stereochemistry at various asymmetric centres has been established by a series of NOED measurements.     

Medium- and light-induced formation of serpentine and anthocyanins in cell suspension cultures of Catharanthus roseus

By irradiation with fluorescent light, medium-induced cell cultures of Catharanthus roseus accumulated anthocyanins and the indole alkaloid serpentine. The formation of both compounds was inhibited by phosphate and nitrogen-containing mineral salts and stimulated by high sucrose concentrations. The accumulation of serpentine was preceded by an increase and subsequent decrease of its biogenetic precursor ajmalicine, which was the predominant alkaloid of medium-induced cultures in the dark. High concentrations of serpentine or anthocyanins were observed only in a small proportion of all cells present in a medium-induced culture. The aglycones of the anthocyanins were identified as petunidin, malvidin and hirsutidin.     

Alkaloid formation by Catharanthus roseus cells in a packed column biofilm reactor

Summary Catharanthus roseus cells producing indole alkaloids were grown on surfaces of Ca-alginate beads within the interspacial volume of a packed column. Production media was circulated through the packed column in an upflow mode. Growth and indole alkaloid formation were quantified and compared with suspension culture of cells. Final alkaloid concentration and alkaloid yield obtained in the packed bed was superior to those obtained in suspension culture. This is thought to be due to improved cell-cell contact and interaction in the packed column.     

Biochemical,immunological and genetic characterization of natural gramine-free variants of Hordeum vulgare L.

Many barley cultivars (e.g. Arimar) contain the indole alkaloid gramine, but some do not. Among seven gramine-free cultivars tested, two phenotypic classes were found: those with a normal level of the N-methyltransferase (NMT) activity that catalyzes the last two steps of gramine synthesis (e.g. Proctor); and those having neither NMT activity nor protein recognized by polyclonal antibodies raised against purified NMT (e.g. Morex).A 3 × 3 diallel cross with reciprocals was made using cultivars Arimar, Proctor and Morex. The pattern of occurrence of gramine and NMT activity among the F₁ hybrids suggested that Proctor and Morex carried defective alleles of the same nuclear gene governing an early step in the indole alkaloid pathway, and that Morex also carried a recessive allele at a nuclear locus encoding NMT activity. However, no non-parental alkaloid phenotypes were found in the F₂ generation from an Arimar × Morex cross and the ratio of progeny with gramine to those with no alkaloids was 3 : 1. One explanation of these results is tight linkage between genes controlling two of the steps in gramine biosynthesis.     

Selection of fungal elicitors to increase indole alkaloid accumulation in catharanthus roseus suspension cell culture

Various fungal elicitors derived from 12 fungi were tested to improve indole alkaloid production in Catharanthus roseus cell suspension cultures. Results show that different fungal mycelium homogenates stimulate different kinds of indole alkaloid (ajmalicine, serpentine and catharanthine) accumulation, which ranged from 2- to 5-fold higher than the control. Some fungal culture filtrates also efficiently elicited the biosynthesis of different indole alkaloids. The optimal elicitor addition and exposure time for the maximal alkaloid production were on day 7 after subculture and for 3 days of treatment but different fungal elicitors showed the different optimal treatment dosages. Additions of elicitor at the doses ranging from 5 mg/l to 30 mg/l of carbon hydrate equivalent resulted in varieous amounts and kinds of indole alkaloid accumulation. Exposed to a same fungal elicitor, several different cell lines generated the different responses regarding as growth rate, culture color and alkaloid production.