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.     

Relationship between cell morphology and indole alkaloid production in suspension cultures of Catharanthus roseus

The relationship between the morphology and indole alkaloid production of Catharanthus roseus cells was investigated. Eleven cell lines were randomly selected from protoplast-derived clones. In each line, most of the cells maintained only one of the two shapes, either spherical or cylindrical. The cell aspect ratio (cell length/width) for most isolates was stable for more than two years of subculture. Cell division patterns of spherical and cylindrical cell isolates were different and patterns of division remained stable in each phenotype and were not considerably affected by auxin or cytokinin levels in the culture media. These observations indicate that cell morphology of our isolates is stable and probably internally determined. Production of the indole alkaloids, ajmalicine and catharanthine was significantly greater when the cell aspect ratio was more than 2.8.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - CPA p-chlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog (1962) medium - SH Schenk and Hildebrandt (1972) medium     

Nutrient consumption and alkaloid accumulation in a hairy root line of Catharanthus roseus

A hairy root line from Catharanthus roseus was cultured in a 14 l bioreactor. Nitrate and phosphate uptakes were similar to the same root line cultured in a 250 ml Erlenmeyer flask. However, sucrose consumption rate was slower in roots cultured in the bioreactor. These results show that it is feasible to upgrade this hairy root line to bioreactor level and, although carbon utilization has to be improved, ajmalicine and catharanthine were still produced and were retained within the biomass tissue.     

Indole alkaloid formation by Catharanthus roseus cells in a biofilm reactor.

Catharanthus roseus cells producing indole alkaloids were grown in the form of a biofilm. Production medium was circulated through the reactor parallel to the upper surface of the horizontal biofilm. Sugar consumption and indole alkaloid formation were followed to compare the performance of cultures with different biofilm thicknesses. Dissolved oxygen concentrations gradients within the biofilms were determined at the end of each run. RNA and protein content of the cells in the upper and lower layers of the the biofilms were compared. Results obtained in the biofilm experiments were compared to those obtained with suspension cultures. At optimized biofilm thicknesses, the biofilm reactor was more effective than suspension cultures in maximizing indole alkaloid titers. This is thought to be due to better cell-cell contact within the biofilm and nutrient concentration gradients, which resulted in low growth rates.     

Influence of auxins on alkaloid accumulation by a transgenic cell line of Catharanthus roseus

The transgenic cell line of Catharanthus roseus (L.) G. Don S10 was used to study the effect of the presence of the synthetic auxins naphthalene acetic acid and 2,4-dichlorophenoxyeacetic acid in the culture medium on the accumulation of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively over-expressed version of the endogenous strictosidine synthase gene. The experiments were carried out using a two-stage culture system, consisting of a growth phase of 7 to 10 days and a production phase of 14 or 30 days. The hormonal composition was a crucial factor when formulating both the growth and the production media. It was determined that the presence of naphthalene acetic acid during the production phase led to lower levels of alkaloid accumulation. The presence of 2,4-dichlorophenoxyacetic acid in the growth medium reduced culture aggregation and repressed secondary metabolism. Cultures grown in medium containing 2,4-dichlorophenoxyacetic acid showed reduced capacity to supply biosynthetic precursors, which resulted in low levels of accumulation of terpenoid indole alkaloids. Cultures grown in 2,4-D-containing medium showed reduced capacity to supply biosynthetic precursors and higher rates of catabolic activity, which resulted in low levels of TIA accumulation. The expression of the gus and strictosidine synthase transgenes, measured at the enzyme level, was similarly high under all conditions tested. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular cloning and analysis of strictosidine beta-D-glucosidase, an enzyme in terpenoid indole alkaloid biosynthesis in Catharanthus roseus

Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to cathenamine. The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.     

Differential regulation of fluorescent alkaloid metabolism between idioblast and lacticifer cells during leaf development in Catharanthus roseus seedlings

Journal of Plant Research - Bioactive specialized (secondary) metabolites are indispensable for plant development or adjustment to their surrounding environment. In many plants, these specialized...     

Fosmidomycin analogues as inhibitors of monoterpenoid indole alkaloid production in Catharanthus roseus cells

Substituted 3-[2-(diethoxyphosphoryl)propyl]oxazolo[4,5-b]pyridine-2(3H)-ones were obtained by functionalization at 6-position with various substituents (aryl, vinyl, carbonyl chains) via reactions catalysed with palladium. We found that these new fosmidomycin analogues inhibited the accumulation of ajmalicine, a marker of monoterpenoid indole alkaloids production in plant cells. Some of them have greater inhibitory effect than fosmidomycin and fully inhibit alkaloid accumulation at the concentration of 100 microM.     

Identification of a low vindoline accumulating cultivar of Catharanthus roseus (L.) G. Don by alkaloid and enzymatic profiling

The Madagascar periwinkle [Catharanthus roseus (L.) G. Don] is a commercially important horticultural flower species and is the only source of the monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, key pharmaceutical compounds used to combat a number of different cancers. The present study uses high performance liquid chromatography for metabolic profiling of the MIAs extracted from seedlings and young leaves of 50 different flowering cultivars of C. roseus to show that, except for a single low vindoline cultivar (Vinca Mediterranean DP Orchid), they accumulate similar levels of MIAs. Further enzymatic studies with extracts from young leaves and from developing seedlings show that the low vindoline cultivar has a 10-fold lower tabersonine-16-hydroxylase activity than those of C. roseus cv. Little Delicata. It is concluded that rapid metabolic and more selective enzymatic profiling of Catharanthus mutants could be useful for the identification of a range of altered MIA biosynthesis lines.     

Two-phase culture for enhanced alkaloid synthesis and release in a new airlift reactor by Catharanthus roseus

A new airlift reactor was used to culture Catharanthus roseus cells, in which the draft tube was made up of polyurethane foam and acted as the immobilizing matrix. The reactor was connected in series to an adsorbent column with a neutral polymeric resin which absorbs these alkaloids. The synthesis of alkaloid was stimulated by adding the resin column and the total content of alkaloid secreted by cells reached 380 mg/L, which was 4.5 times of that in the control experiment. Meanwhile, most of the intracellular alkaloid produced by Catharanthus roseus was secreted into the medium.     

Large scale fermentation and alkaloid production of cell suspension cultures of Catharanthus roseus

Cell cultures of Catharanthus roseus were scaled up to volumes of 50001 using conventional reactors equipped with flat-blade impellers. The behavior of the fermenter grown cells was compared with corresponding shake flask experiments with respect to growth and indole alkaloid inducibility and production. The limits and problems of transferring shake flask experiments of culture systems such as Catharanthus, in which alkaloid production depends greatly upon the physiological state of the cells, to large scale multistage processes is discussed.     

Abnormalities in carbohydrate and lipid metabolisms in high-fructose dietfed insulin-resistant rats: amelioration by Catharanthus roseus treatments

High intake of dietary fructose has been shown to exert a number of adverse metabolic effects in humans and experimental animals. The present study was proposed to elucidate the effect of Catharanthus roseus (C. roseus) leaf powder treatment on alterations in carbohydrate and lipid metabolisms in rats fed with high-fructose diet. Male Wistar rats of body weight around 180 g were divided into four groups, two of these groups (groups C and C+CR) were fed with standard pellet diet and the other two groups (groups F and F+CR) were fed with high-fructose (66 %) diet. C. roseus leaf powder suspension in water (100 mg/kg body weight/day) was administered orally to group C+CR and group F+CR. At the end of a 60-day experimental period, biochemical parameters related to carbohydrate and lipid metabolisms were assayed. C. roseus treatment completely prevented the fructose-induced increased body weight, hyperglycemia, and hypertriglyceridemia. Hyperinsulinemia and insulin resistance observed in group F was significantly decreased with C. roseus treatment in group F+CR. The alterations observed in the activities of enzymes of carbohydrate and lipid metabolisms and contents of hepatic tissue lipids in group F rats were significantly restored to near normal values by C. roseus treatment in group F+CR. In conclusion, our study demonstrates that C. roseus treatment is effective in preventing fructose-induced insulin resistance and hypertriglyceridemia while attenuating the fructose-induced alterations in carbohydrate and lipid metabolisms. This study suggests that the plant can be used as an adjuvant for the prevention and/or management of insulin resistance and disorders related to it.     

Improvement of indole alkaloid production in Catharanthus roseus cell cultures by osmotic shock

Catharanthine production in Catharanthus roseussuspension cell cultures was increased by about 4-fold to 28 mg l^–1, 23 mg l^–1and 24 mg l^–1by adding sodium alginate, mannitol or polyvinyl pyrrolidone, respectively. Sodium alginate and polyvinyl pyrrolidone also enhanced ajmalicine production to 28 mg l^–1and 31 mg l^–1, respectively. Up to 55–70% of the total alkaloids were released into the medium. These treatments could stimulate higher alkaloid production in C. roseuscell cultures than NaCl and KCl stresses. The possible mechanisms for these treatment effects are discussed.     

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.     

Effects of buffered media upon growth and alkaloid production of Catharanthus roseus hairy roots

The influence of buffered media upon the growth and alkaloid productivity of Catharanthus roseus hairy root culture was examined. As expected, the buffers minimized shifts in the pH of the media and had slightly negative effects upon growth. The growth of the hairy roots remained optimal in unbuffered media. The specific yield of lochnericine was significantly lower in response to the addition of buffers, while tabersonine was significantly higher. In contrast, the specific yields of ajmalicine, serpentine, and horhammericine remained unchanged.     

盐胁迫对长春花幼苗生长和生物碱含量的影响

以NaCl浓度分别为0、50、100、150、200和250 mmol·L^-1的1/2 Hoagland营养液处理长春花幼苗,7 d后测定其鲜质量、干质量、丙二醛(MDA)和叶绿素含量、色氨酸脱羧酶（TDC）和过氧化物酶（POD）活性等生理指标及文多灵、长春质碱、长春新碱和长春碱等生物碱含量.结果表明：NaCl显著地降低长春花幼苗的鲜质量和干质量,提高MDA含量;叶绿素含量在低盐浓度(50 mmol·L^-1)下与对照相比差异不显著,在高于50 mmol·L^-1时随NaCl浓度的增加而逐渐降低;在NaCl处理下,POD活性与对照相比显著上升;TDC活性在50 mmol·L^-1 NaCl处理下活性最高,而后随盐浓度的增加逐渐降低;文多灵、长春质碱、长春新碱和长春碱含量都是在50 mmol·L^-1NaCl处理下最高,分别为4.61、3.56、1.19和2.95 mg·g^-1,并显著高于对照及其他处理.盐胁迫虽然在一定程度上抑制了长春花幼苗生长,但促进了其生物碱的代谢,提高了生物碱含量;50 mmol·L^-1NaCl处理对长春花吲哚生物碱代谢的促进作用最大.