Involvement of nitric oxide signaling in mammalian Bax-induced terpenoid indole alkaloid production of Catharanthus roseus cells

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, has been demonstrated to be a potential regulatory factor for plant secondary metabolite biosynthesis recently. To investigate the molecular mechanism of Bax-induced secondary metabolite biosynthesis, we determined the contents of nitric oxide (NO) of the transgenic Catharanthus roseus cells overexpressing a mouse Bax protein and checked the effects of NO specific scavenger 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide (cPITO) on Bax-induced terpenoid indole alkaloid (TIA) production of the cells. The data showed that overexpression of the mouse Bax in C. roseus cells triggered NO generation of the cells. Treatment of cPITO not only inhibited the Bax-triggered NO burst but also suppressed the Bax-induced TIA production. The results indicated that the mouse Bax might activate the NO signaling in C. roseus cells and induce TIA production through the NO-dependent signal pathway in the cells. Furthermore, the activities of nitric oxide synthase (NOS) were significantly increased in the transgenic Bax cells as compared to those in the control cells, showing that the mouse Bax may induce NOS of C. roseus cells. Treatment of the transgenic Bax cells with NOS inhibitor PBITU blocked both Bax-induced NO genera- tion and TIA production, which suggested that the mouse Bax might trigger NO generation and TIA production through NOS. However, the NOS-like activities and NO generation in the transgenic Bax cells did not match kinetically and the Bax-induced NOS-like activity was much later and lower than NO production. Moreover, the Bax-induced NO generation and TIA production were only partially inhibited by PBITU. Thus, our results suggested that the Bax-induced NO production and secondary metabolite biosynthesis in C. roseus cells was not entirely dependent on NOS or NOS-like enzymes.     

Somatic embryogenesis and indole alkaloid production in Catharanthus roseus

ABSTRACT

A new protocol to obtain an embryogenic cell line from cultured seedling explants of Catharanthus roseus is described. In order to assess the relationship between tissue differentiation and secondary metabolite biosynthesis, the biosynthetic capabilities (alkaloid production) of an embryogenic cell line and two non-embryogenic C. roseus strains were comparatively examined. Faster cell growth rate was associated with higher alkaloid production in the embryogenic cell line. The kinetics of ajmalicine and serpentine production by the three cell lines is also reported.     

Compact callus cluster suspension cultures of Catharanthus roseus with enhanced indole alkaloid biosynthesis

Summary Compact callus clusters showing a certain level of cellular or tissue differentiation were established from Catharanthus roseus stem and leaf explants in a modified MS liquid induction medium supplemented with 5.37 μM α-naphthaleneacetic acid and 4.65 μM kinetin. In the induction medium most leaf explants developed into friable half-closed hollow callus clusters, whereas in the same medium containing 2,4-dichlorophenoxyacetic acid instead of α-naphthaleneacetic acid, most leaf explants were induced to form dispersed cell suspension cultures. Characteristics of these different types of suspension cultures were compared, and the results showed that the compact callus clusters could synthesize indole alkaloids 1.9- and 2.4-fold higher than the half-closed hollow callus clusters and dispersed cell cultures, respectively. The degree of compaction expressed by the ratio of fresh weight to dry weight of these suspension cultures was correlated to indole alkaloid production. Our studies also postulated that the level of cellular/tissue differentiation might be responsible for these different alkaloid synthesis capabilities. Sucrose regime affected some properties (the size, degree of compaction, differentiation level) of the compact callus cluster cultures and therefore influenced alkaloid production.     

Effects of PEG-induced drought stress on regulation of indole alkaloid biosynthesis in Catharanthus roseus

Catharanthus roseus (C. roseus) plants were used to investigate the terpenoid indole alkaloids (TIAs) accumulation under the condition of PEG-induced drought stress. Multivariate analysis showed that 35% PEG6000 could induce more obvious and stable accumulation on proline (PRO) content and the relative water content (RWC). The results indicated that there were gradual increase and then decrease (p?<?.05) in the contents of vindoline (VIN) and catharanthine (CAT) under 35% PEG6000 stress, but the content of vinblastine (VBL) increased gradually. In addition, the expression levels of tryptophan decarboxylase (TDC), strictosidine synthase (STR) and deacetylvindoline-4-O-acetyltransferase (DAT) were upregulated in plants under 35% PEG6000 stress. Further correlation analysis indicated that CAT accumulation was significantly correlated with TDC gene expression, and VBL accumulation was significantly correlated with peroxidase (p?<?.05). Our results suggest that the cultivation of C. roseus in drought stress would serve as effective treatment for accumulating TIAs.     

The role of strictosidine in monoterpenoid indole alkaloid biosynthesis

In contrast to previous reports that vincoside was the sole precursor for indole alkaloids in Vinca rosea, the 3α epimer strictosidine has been incorporated into tetrahydroalstonine, ajmalicine, catharanthine and vindoline; the anomalous 3β to 3α inversion is no longer required.     

Catharanthus terpenoid indole alkaloids: biosynthesis and regulation

Catharanthus roseus is still the only source for the powerful antitumour drugs vinblastine and vincristine. Some other pharmaceutical compounds from this plant, ajmalicine and serpentine are also of economical importance. Although C. roseus has been studied extensively and was subject of numerous publications, a full characterization of its alkaloid pathway is not yet achieved. Here we review some of the recent work done on this plant. Most of the work focussed on early steps of the pathway, particularly the discovery of the 2-C-methyl-d-erythritol 4-phosphate (MEP)-pathway leading to terpenoids. Both mevalonate and MEP pathways are utilized by plants with apparent cross-talk between them across different compartments. Many genes of the early steps in Catharanthus alkaloid pathway have been cloned and overexpressed to improve the biosynthesis. Research on the late steps in the pathway resulted in cloning of several genes. Enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their localization and regulation are discussed. Much progress has been made at alkaloid regulatory level. Feeding precursors, growth regulators treatments and metabolic engineering are good tools to increase productivity of terpenoid indole alkaloids. But still our knowledge of the late steps in the Catharanthus alkaloid pathway and the genes involved is limited.     

Substrate specificity and diastereoselectivity of strictosidine glucosidase, a key enzyme in monoterpene indole alkaloid biosynthesis

Strictosidine glucosidase (SGD) from Catharanthus roseus catalyzes the deglycosylation of strictosidine, an intermediate from which thousands of monoterpene indole alkaloids are derived. The steady-state kinetics of SGD with a variety of strictosidine analogs revealed the substrate preferences of this enzyme at two key positions of the strictosidine substrate. Additionally, SGD from C. roseus turns over both strictosidine and its stereoisomer vincoside, indicating that although this enzyme prefers the naturally occurring diastereomer, the enzyme is not completely diastereoselective. The implications of the substrate specificity of SGD in metabolic engineering efforts of C. roseus are highlighted.     

3D-Structure and function of strictosidine synthase--the key enzyme of monoterpenoid indole alkaloid biosynthesis

Strictosidine synthase (STR; EC 4.3.3.2) plays a key role in the biosynthesis of monoterpenoid indole alkaloids by catalyzing the Pictet-Spengler reaction between tryptamine and secologanin, leading exclusively to 3alpha-(S)-strictosidine. The structure of the native enzyme from the Indian medicinal plant Rauvolfia serpentina represents the first example of a six-bladed four-stranded beta-propeller fold from the plant kingdom. Moreover, the architecture of the enzyme-substrate and enzyme-product complexes reveals deep insight into the active centre and mechanism of the synthase highlighting the importance of Glu309 as the catalytic residue. The present review describes the 3D-structure and function of R. serpentina strictosidine synthase and provides a summary of the strictosidine synthase substrate specificity studies carried out in different organisms to date. Based on the enzyme-product complex, this paper goes on to describe a rational, structure-based redesign of the enzyme, which offers the opportunity to produce novel strictosidine derivatives which can be used to generate alkaloid libraries of the N-analogues heteroyohimbine type. Finally, alignment studies of functionally expressed strictosidine synthases are presented and the evolutionary aspects of sequence- and structure-related beta-propeller folds are discussed.