Anna Elisabeth Schulte

Mevalonate kinase activity in Catharanthus roseus plants and suspension cultured cells.

Mevalonate kinase is an early enzyme in plant isoprenoid biosynthesis. Its activity was studied in different parts of Catharanthus roseus (L.) G. Don (Apocynaceae) plants and in C. roseus suspension cultured cells. In the plant specific mevalonate kinase activities were found to be relatively high in the fruits, stem, roots, flowers and buds, and relatively low in young and completely elongated leaves. In suspension cultured cells, the specific mevalonate kinase activity increased during the exponential phase of growth, reaching a maximum of 0.5 nkat/mg protein at 4 days after subculturing. After 6 days, which corresponded to the beginning of the stationary phase, the specific activity decreased. After transferring 14-day-old C. roseus cells to a medium known to induce the production of terpenoid indole alkaloids, an increase in the specific mevalonate kinase activity was observed, reaching a maximum of 1.7 nkat/mg protein at 8 days after transfer. The alkaloid accumulation in the cultures was monitored, and ajmalicine was found to be the major product. Under standard conditions, mevalonate kinase activity showed a diurnal rhythm, with highest activities at 12:00 and 24:00.

Purification and characterization of phosphomevalonate kinase from Catharanthus roseus

Abstract 5-Phosphomevalonate kinase activity was partially purified from suspension cultured cells of Catharanthus roseus (L.) G. Don. The enzyme had an estimated M r of 128,000 as determined by size-exclusion chromatography. Kinetic studies indicated that the mechanism of action was sequential with true K m values of 0.35 and 0.22 mM for 5-phosphomevalonate and ATP, respectively. The enzyme activity was dependent on the presence of divalent ions with a preference for Mg 2+ ; at equimolar concentrations, i.e., 2 mM, the enzyme showed a relative remaining activity of 52, 22 and 4% in the presence of Mn 2+ , Zn 2+ and Ni 2+ , respectively (Mg 2+ =100%). The pH optimum for 5-phosphomevalonate kinase activity ranged from 7 to 9, with a maximum at about 8.

Investigation of the Chemomarkers Correlated with Flower Colour in Different Organs of Catharanthus roseus Using NMR‐based Metabolomics

INTRODUCTION Flower colour is a complex phenomenon that involves a wide range of secondary metabolites of flowers, for example phenolics and carotenoids as well as co-pigments. Biosynthesis of these metabolites, though, occurs through complicated pathways in many other plant organs. The analysis of the metabolic profile of leaves, stems and roots, for example, therefore may allow the identification of chemomarkers related to the final expression of flower colour. OBJECTIVE To investigate the metabolic profile of leaves, stems, roots and flowers of Catharanthus roseus and the possible correlation with four flower colours (orange, pink, purple and red). METHODS (1) H-NMR and multivariate data analysis were used to characterise the metabolites in the organs. RESULTS The results showed that flower colour is characterised by a special pattern of metabolites such as anthocyanins, flavonoids, organic acids and sugars. The leaves, stems and roots also exhibit differences in their metabolic profiles according to the flower colour. Plants with orange flowers featured a relatively high level of kaempferol analogues in all organs except roots. Red-flowered plants showed a high level of malic acid, fumaric acid and asparagine in both flowers and leaves, and purple and pink flowering plants exhibited high levels of sucrose, glucose and 2,3-dihydroxy benzoic acid. High concentrations of quercetin analogues were detected in flowers and leaves of purple-flowered plants. CONCLUSIONS There is a correlation between the metabolites specifically associated to the expression of different flower colours and the metabolite profile of other plant organs and it is therefore possible to predict the flower colours by detecting specific metabolites in leaves, stems or roots. This may have interesting application in the plant breeding industry.

Characterization of some isoprenoid-biosynthetic enzymes from plant cell cultures

Publisher Summary This chapter presents characterization of some isoprenoid–biosynthetic enzymes from plant cell cultures. Plant secondary metabolites are biosynthesized from simple building blocks supplied by primary metabolism. Two important metabolic routes include the shikimate pathway and the isoprenoid biosynthesis. The shikimate pathway leads to the synthesis of phenolic compounds and the aromatic amino acids phenylalanine, tyrosine, and tryptophan. The isoprenoid biosynthesis is a heavily branched pathway leading to a broad spectrum of compounds. Plant enzymes can often be extracted with more ease and better yields from plant cell cultures as compared to the intact plants. Acetoacetyl-CoA thiolase catalyses the reversible condensation of 2 molecules of acetyl-CoA yielding acetoacetyl-CoA and CoASH. The spectrophotometric assay is based on the monitoring of the, CoASH depending, rate of consumption of acetoacetyl-CoA at 300 nm. 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase couples another acetyl-CoA molecule to acetoacetyl-CoA, yielding HMG-CoA and CoASH. The enzyme is irreversibly inhibited by L-659,699, a metabolite known to inhibit mammalian HMG-CoA synthase specifically, indicating a similar catalytic mechanism for the plant enzyme.

Metabolic alteration of Catharanthus roseus cell suspension cultures overexpressing geraniol synthase in the plastids or cytosol

Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering.

Erratum to: Analysis of metabolites in the terpenoid pathway of Catharanthus roseus cell suspensions

In Catharanthus roseus cell cultures, the monoterpenoid pathway has been shown to be a limiting factor in terpenoid indole alkaloid (TIA) production. This could be due to competition at the level of isopentenyl diphosphate::dimethylallyl diphosphate (C5) which leads to the biosynthesis of different terpenoid groups. For future engineering of the terpenoid pathway, chemical characterization of C. roseus cell cultures is a necessity. Therefore, in this study nine C. roseus cell suspension lines were characterized by analyzing the levels of the major terpenoids derived from different biosynthetic pathways which may compete for the same precursors; TIA (monoterpenoid, C10), carotenoids (tetraterpenoid, C40), and sterols (triterpenoid, C30). Among the cell lines, CRPP (S) was the most promising TIA-producing cell line which provided more TIA [24 lmol g dry weight (DW)] than carotenoids (15 lmol g DW) and sterols (2 lmol g DW). However, when considering the distribution of the isopentenylprecursor (C5), the carotenoids which assemble from 89 C5 represent twofold more C5-units (122 lmol g DW) than the TIA in this cell line. In the CRPP (G), A12A2 (G), and A12A2 (S) cell lines, the C5 distribution was predominant toward carotenoid biosynthesis as well, resulting in a relatively high accumulation of carotenoids. The geranylgeranyl diphosphate (C20) pathway toward carotenoid production is therefore considered competitive toward TIA biosynthesis. For channeling more precursors to the TIA, the branch point for C10 and C20 seems an interesting target for metabolic engineering. Using principal component analysis of the chromatographic data, we characterized the cell lines chemically based on their metabolite levels. The information on the metabolic composition of C. roseus cell cultures is useful for developing strategies to engineer the metabolic pathways and for selection of cell lines for future studies.