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.     

Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis.

Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti-cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.     

A novel jasmonate- and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitor-inducible AP2-domain transcription factor, ORCA2.

Jasmonate (JA) is an important plant stress hormone that induces various plant defense responses, including the biosynthesis of protective secondary metabolites. The induction of the secondary metabolite biosynthetic gene Strictosidine synthase (Str) in Catharanthus roseus (periwinkle) cells by elicitor requires JA as a second messenger. A 42 bp region in the Str promoter is both necessary and sufficient for JA- and elicitor-responsive expression. This region is unlike other previously identified JA-responsive regions, and contains a GCC-box-like element. Yeast one-hybrid screening identified cDNAs encoding two AP2-domain proteins. These octadecanoid-derivative responsive Catharanthus AP2-domain (ORCA) proteins bind in a sequence-specific manner the JA- and elicitor-responsive element. ORCA2 trans-activates the Str promoter and its expression is rapidly inducible with JA and elicitor, whereas Orca1 is expressed constitutively. The results indicate that a GCC-box-like element and ORCA2 play key roles in JA- and elicitor-responsive expression of the terpenoid indole alkaloid biosynthetic gene Str.     

Involvement of nitric oxide signaling in mammalian Bax-induced terpenoid indole alkaloid production of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> 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 generation 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.     

Screening method for cDNAs encoding putative enzymes converting loganin into secologanin by a transgenic yeast culture

A screening method was developed for the detection of enzymes converting loganin to secologanin, a precursor in the biosynthesis of indole alkaloids. The method uses a transgenic yeast culture expressing two cDNAs encoding enzymes involved in the terpenoid indole alkaloid biosynthesis. In the presence of secologanin, the yeast culture produces a yellow compound visible on nitrocellulose. This color change was used to screen a cDNA library of Catharanthus roseus for a putative enzyme converting loganin into secologanin.     

Opium poppy and Madagascar periwinkle: model non-model systems to investigate alkaloid biosynthesis in plants

Alkaloids represent a large and diverse group of compounds that are related by the occurrence of a nitrogen atom within a heterocyclic backbone. Unlike other types of secondary metabolites, the various structural categories of alkaloids are unrelated in terms of biosynthesis and evolution. Although the biology of each group is unique, common patterns have become apparent. Opium poppy ( Papaver somniferum ), which produces several benzylisoquinoline alkaloids, and Madagascar periwinkle ( Catharanthus roseus ), which accumulates an array of monoterpenoid indole alkaloids, have emerged as the premier organisms used to study plant alkaloid metabolism. The status of these species as model systems results from decades of research on the chemistry, enzymology and molecular biology responsible for the biosynthesis of valuable pharmaceutical alkaloids. Opium poppy remains the only commercial source for morphine, codeine and semi-synthetic analgesics, such as oxycodone, derived from thebaine. Catharanthus roseus is the only source for the anti-cancer drugs vinblastine and vincristine. Impressive collections of cDNAs encoding biosynthetic enzymes and regulatory proteins involved in the formation of benzylisoquinoline and monoterpenoid indole alkaloids are now available, and the rate of gene discovery has accelerated with the application of genomics. Such tools have allowed the establishment of models that describe the complex cell biology of alkaloid metabolism in these important medicinal plants. A suite of biotechnological resources, including genetic transformation protocols, has allowed the application of metabolic engineering to modify the alkaloid content of these and related species. An overview of recent progress on benzylisoquinoline and monoterpenoid indole alkaloid biosynthesis in opium poppy and C. roseus is presented.     

药用植物长春花WRKY转录因子的鉴定及表达谱分析

WRKY是调控植物生长发育和逆境胁迫反应等生命活动的一个转录因子大家族.然而,有关药用植物长春花CrWRKY转录因子的种类和功能却知之甚少.从26 009个长春花蛋白中鉴定出47个CrWRKY转录因子.依据WRKY结构域和系统进化,将CrWRKY分为G1、G2和G3三大类群.表达谱数据分析表明,长春花CrWRKY基因的表达具有器官特异性.47个CrWRKY基因的表达谱可分为3种表达模式:第1类型主要在花、甲基茉莉酸甲酯(MeJA)或酵母提取物(YE)处理的原生质体中高表达;第2类型主要在茎和毛状根中高表达;第3类型在根、茎、叶、幼苗和MeJA处理的毛状根中高表达.进一步用实时定量PCR检测了16个代表性CrWRKY基因在不同器官、MeJA处理原生质体和毛状根中的表达模式,检测结果与上述数字基因表达谱数据基本一致.约1/3以上CrWRKY基因的表达受MeJA和YE的调控,暗示它们可能参与萜类吲哚生物碱的合成和逆境胁迫反应.为进一步解析长春花WRKY转录因子的功能和萜类吲哚生物碱合成调控的网络奠定了基础.     

Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over-expressing the alpha or the alpha and beta subunits of anthranilate synthase

Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in h?rhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.     

Quantification of metabolites in the indole alkaloid pathways of catharanthus roseus: implications for metabolic engineering

In this article, we present a review of the current state of metabolic engineering in Catharanthus roseus. A significant amount of research has contributed to characterization of several individual steps in the biosynthetic pathway of medicinally valuable alkaloids. However, knowledge of the regulation of these pathways is still sparse. Using hairy root cultures, we studied the responses of alkaloid metabolism to environmental stimulation such as light and elicitation. Through precursor feeding studies, the putative rate-limiting steps of the terpenoid pathway in hairy root cultures also have been examined. Relating this knowledge to specific events at the molecular level, and the cloning of corresponding genes are the next key steps in metabolic engineering of the C. roseus alkaloids. Copyright 1998 John Wiley & Sons, Inc.     

Cloning, characterization and localization of a novel basic peroxidase gene from Catharanthus roseus

Catharanthus roseus (L.) G. Don produces a number of biologically active terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic pathway. The final dimerization step of this pathway, leading to the synthesis of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic peroxidase. However, reports of the gene encoding this enzyme are scarce for C. roseus. We report here for the first time the cloning, characterization and localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp partial peroxidase cDNA (CrInt1) was initially amplified from the internodal stem tissue, using degenerate oligonucleotide primers, and cloned. The full-length coding region of CrPrx cDNA was isolated by screening a leaf-specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence encodes a deduced translation product of 330 amino acids with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa, and the pI value is 8.68. CrPrx was found to belong to a 'three intron' category of gene that encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were found to be present in specific organs and were regulated by different stress treatments. Using a beta-glucuronidase-green fluorescent protein fusion of CrPrx protein, we demonstrated that the fused protein is localized in leaf epidermal and guard cell walls of transiently transformed tobacco. We propose that CrPrx is involved in cell wall synthesis, and also that the gene is induced under methyl jasmonate treatment. Its potential involvement in the terpenoid indole alkaloid biosynthetic pathway is discussed.     

Transcript profiling of terpenoid indole alkaloid pathway genes and regulators reveals strong expression of repressors in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> cell cultures

The understanding of the complexities and molecular events regulating genes and the activators involved in terpenoid indole alkaloid (TIA) metabolism is known to a certain extent in cell cultures of an important TIA yielding plant, Catharanthus roseus, though it is not yet complete. Recently, the repressors of early TIA pathway genes have also been identified. However, their roles in the regulation of TIA pathway in C. roseus cell cultures remains yet unknown. We have made a comparative profiling of genes catalyzing the important steps of 2-C methyl-D-erythritol-4-phosphate (MEP), shikimate and TIA biosynthetic pathways, their activator and repressors using macroarray, semiquantitative RT-PCR and northern analyses in a rotation culture system of C. roseus comprising differentiated and proliferated cells. Our results demonstrate that TIA biosynthetic pathway genes and their activators show variable expression pattern, which was correlated with the changes in the cellular conditions in these systems. Under similar conditions, TIA pathway repressors show strong and consistent expression. The role of repressors in the complex regulation of the TIA pathway in C. roseus cell cultures is discussed. The results were supported by HPLC data, which demonstrated that the molecular program of cellular differentiation is intimately linked with TIA pathway gene expression and TIA production in C. roseus cell cultures.