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Qian Chen,
Zunwei Chen,
Li Lu,
Haihong Jin,
Lina Sun,
Qin Yu,
Hongke Xu,
Fengxia Yang,
Mengna Fu,
Shengchao Li,
Huizhong Wang, Maojun Xu
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ABSTRACT: Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor-induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up-regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90-induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor-induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up-regulation of Str and Tdc. ABA-induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO-induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90-induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90-induced catharanthine biosynthesis of C. roseus cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog.,, 2013.
Biotechnology Progress 04/2013; · 2.34 Impact Factor
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ABSTRACT: The effects of nitric oxide (NO) on antioxidant activity and contents of phenolics and flavonoids in mushroom Russula griseocarnosa were investigated. Freshly harvested mushrooms were fumigated with 0, 10, 20 and 30μLL(-1) NO at 20°C for 2h and then taken to examine the antioxidant activities using assays of reducing power, chelating effect on ferrous ions, scavenging effect on hydroxyl free radicals, and 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity. The results showed that the antioxidant activities of the mushrooms fumigated with NO were significantly increased when compared to the controls. Moreover, NO fumigation significantly enhanced phenolic and flavonoid contents and stimulated the activities of phenylalanine ammonia-lyase and chalcone synthase. The results indicated that NO fumigation might have potential application for enhancing the bioactive compounds and improving antioxidant activities in the mushrooms. Furthermore, the data suggested that the NO-induced phenolic and flavonoid accumulation was due to the activation of the biosynthetic pathways in the mushrooms.
Food Chemistry 12/2012; 135(3):1220-5. · 3.65 Impact Factor
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ABSTRACT: PB90 is a novel protein elicitor isolated from Phytophthora boehmeriae. Here, we report that treatment of PB90 stimulates hypericin production and hydrogen peroxide (H2O2) generation in Hypericum perforatum L. cells and demonstrate that H2O2 is essential for PB90-induced hypericin production. To further study the source of PB90-triggered H2O2, we have investigated activities of plasma membrane NADPH oxidase in Hypericum perforatum L. cells subjected to PB90 treatment. It is revealed that treatment of the cells with PB90 significantly increases NADPH
oxidase activity. NADPH oxidase inhibitors suppress not only the PB90-stimulated NADPH oxidase activity but also the PB90-triggered
H2O2 generation and PB90-induced hypericin production, showing that NADPH oxidase is involved in PB90-triggered H2O2 generation and hypericin production. Moreover, the suppression of NADPH oxidase inhibitors on PB90-induced hypericin production
can be reversed by H2O2, although H2O2
per se has no effects on hypericin production of the cells. Together, the data demonstrate that PB90 may induce hypericin production
of H. perforatum cells through the NADPH oxidase-mediated H2O2 signaling pathway.
KeywordsHypericin–PB90–H2O2
–NADPH oxidase–
H. perforatum cells
Plant Cell Tissue and Organ Culture 04/2012; 105(1):47-53. · 3.09 Impact Factor
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ABSTRACT: Elicitor prepared from the cell walls ofPenicillium citrinum induces multiple responses ofTaxus chinensis cells, including nitric oxide (NO) generation, sequentially followed by the activation of PAL and synthesis of taxol. NO
scavenger cPITO and nitric oxide synthase (NOS) inhibitor PBITU prevent the latter two reactions, all of which are triggered
in the absence of elicitor by NO donor sodium nitroprusside (SNP). The elicitor-induced NO release ofTaxus chinensis suspension cells is strongly inhibited by PBITU. These results demonstrate a causal relationship between NO generation and
the latter two reactions ofTaxus chinensis cells to the elicitor, and also indicate that NO, produced via NOS inTaxus chinensis cells treated with fungal elicitor, might act as an essential signaling molecule for triggering the activation of PAL and
synthesis of taxol.
Keywordsnitric oxide-elicitor-PAL-
Taxus chinensis cell-taxol
Chinese Science Bulletin 04/2012; 49(10):1038-1043. · 1.32 Impact Factor
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ABSTRACT: Exposure to ozone induced a rapid increase in the levels of the sesquiterpene phytohormone abscisic acid (ABA) and the isoflavone puerarin in suspension cell cultures of Pueraria thomsnii Benth. The observed increases in ABA and puerarin were dependent on the concentration of ozone applied to P. thomsnii cell cultures. In order to examine the role of ABA in ozone-induced puerarin production, cell suspensions were pretreated with the ABA biosynthetic inhibitor fluridone. Following ozone exposure, fluridone treatment suppressed ABA accumulation suggesting ABA was normally synthesized de novo through the carotenoid pathway. Fluridone also blocked ozone-induced puerarin production, which could be reversed through application of exogenous ABA. However, in the absence of ozone, ABA itself had no effect on puerarin accumulation in the suspension cells. Taken together, the data indicate that ozone is an efficient elicitor of puerarin production and may be particularly applicable for improving puerarin production in plant cell cultures. Furthermore, we demonstrate that ABA is one factor associated with ozone-induced puerarin production in P. thomsnii cell cultures.
Plant Cell Reports 09/2011; 31(1):179-85. · 2.27 Impact Factor
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ABSTRACT: Exposure to ozone induced a rapid increase in the levels of the phytohormone abscisic acid (ABA) and sequentially followed by the enhancement of Taxol production in suspension cell cultures of Taxus chinensis. The observed increases in ABA and Taxol were dependent on the concentration of ozone applied to T. chinensis cell cultures. To examine the role of ABA in ozone-induced Taxol production, we pretreated the cells with ABA biosynthesis inhibitor fluridone to abolish ozone-triggered ABA generation and assayed the effect of fluridone on ozone-induced Taxol production. The results showed that pretreatment of the cells with fluridone not only suppressed the ozone-triggered ABA generation but also blocked the ozone-induced Taxol production. Moreover, our data indicate that the effect of ABA on Taxol production of T. chinensis cell cultures is dose-dependent. Interestingly, the suppression of fluridone on ozone-induced Taxol production was reversed by exogenous application of low dose of ABA, although treatment of low dose ABA alone had no effect on Taxol production of the cells. Together, the data indicated that ozone was an efficient elicitor for improving Taxol production of plant cell cultures. Furthermore, we demonstrated that ABA played critical roles in ozone-induced Taxol production of T. chinensis suspension cell cultures.
Biotechnology Progress 06/2011; 27(5):1415-20. · 2.34 Impact Factor
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ABSTRACT: Accumulation of secondary metabolites is one of the common reactions of plants to ozone exposure in nature. To investigate the effect of ozone on the production of desired compounds of plant cell cultures, we assayed hypericin production of Hypericum perforatum suspension cell cultures treated with different doses of ozone at different culture phases. The results show that hypericin contents of the cells treated with 60 to 180 nL L(-1) ozone are significantly higher than those of the control, showing that ozone exposure may stimulate hypericin synthesis. Hypericin production of the cells treated with ozone at exponential phase is higher than that of lag and stationary phase, which suggests that exponential phase cell cultures are more responsive to ozone exposure than lag and stationary phase cells. The highest hypericin production is obtained by the cells exposed to 90 nL L(-1) ozone at late exponential phase for 3 h, being about fourfold of the control. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.
Biotechnology Progress 03/2011; · 2.34 Impact Factor
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ABSTRACT: Secondary metabolite accumulation and nitric oxide (NO) generation are two common responses of plant cells to fungal elicitors, and NO has been reported to play important roles in elicitor-induced secondary metabolite production. However, the source of elicitor-triggered NO generation in plant cells remains largely unknown. To investigate the origin of elicitor-triggered NO, we examined nitrate reductase (NR) activities and the expression levels of NIA1 and NIA2 genes of Camptotheca acuminata cells treated with PB90, a protein elicitor from Phytophthora boehmeriae. The data show that PB90 treatment stimulates NR activity and induces upregulation of NIA1 but does not affect NIA2 expression in the cells. Pretreatment of the cells with NR inhibitors tungstate and Gln abolishes not only the fungal elicitor-triggered NR activities but also the PB90-induced NO generation. Treatment of PB90 enhances camptothecin contents of the cells, suggesting that the fungal elicitor might stimulate camptothecin biosynthesis. Furthermore, application of tungstate and Gln suppresses the fungal elicitor-induced camptothecin accumulation of the cells and the suppression of NR inhibitors on PB90-induced camptothecin production can be reversed by NO via its donor sodium nitroprusside. Together, the results suggest that NIA1 is sensitive to PB90 and the fungal elicitor-induced upregulation of NIA1 may lead to higher NR activity. Furthermore, our data demonstrate that NR is involved in the fungal elicitor-triggered NO generation and the fungal elicitor induces camptothecin production of C. acuminata cells dependently on NR-mediated NO generation.
Applied Microbiology and Biotechnology 02/2011; 90(3):1073-81. · 3.42 Impact Factor
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ABSTRACT: Heat shock (HS, 40 degrees C, 10 min) induces hypericin production, nitric oxide (NO) generation, and hydrogen peroxide (H(2)O(2)) accumulation of Hypericum perforatum suspension cells. Catalase (CAT) and NO specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) suppress not only the HS-induced H(2)O(2) generation and NO burst, but also the HS-triggered hypericin production. Hypericin contents of the cells treated with both NO and H(2)O(2) are significantly higher than those of the cells treated with NO alone, although H(2)O(2) per se has no effects on hypericin production of the cells, which suggests the synergistic action between H(2)O(2) and NO on hypericin production. NO treatment enhances H(2)O(2) levels of H. perforatum cells, while external application of H(2)O(2) induces NO generation of cells. Thus, the results reveal a mutually amplifying action between H(2)O(2) and NO in H. perforatum cells. CAT treatment inhibits both HS-induced H(2)O(2) accumulation and NO generation, while cPTIO can also suppress H(2)O(2) levels of the heat shocked cells. The results imply that H(2)O(2) and NO may enhance each other's levels by their mutually amplifying action in the heat shocked cells. Membrane NAD(P)H oxidase inhibitor diphenylene iodonium (DPI) and nitric oxide synthase (NOS) inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU) not only inhibit the mutually amplifying action between H(2)O(2) and NO but also abolish the synergistic effects of H(2)O(2) and NO on hypericin production, showing that the synergism of H(2)O(2) and NO on secondary metabolite biosynthesis might be dependent on their mutual amplification. Taken together, data of the present work demonstrate that both H(2)O(2) and NO are essential for HS-induced hypericin production of H. perforatum suspension cells. Furthermore, the results reveal a special interaction between the two signal molecules in mediating HS-triggered secondary metabolite biosynthesis of the cells.
Science in China Series C Life Sciences 09/2008; 51(8):676-86. · 1.61 Impact Factor
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ABSTRACT: 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.
Science in China Series C Life Sciences 01/2008; 50(6):799-807. · 1.61 Impact Factor
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ABSTRACT: Bax, a mammalian pro-apoptotic member of the Bcl-2 family, triggers hypersensitive reactions when expressed in plants. To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells, we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the beta-estradiol-inducible promoter. The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on beta-estradiol concentrations applied. Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 micromol/L beta-estradiol are 5.0-and 5.5-fold of the control cells. Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str, two key genes in terpenoid indole alkaloid biosynthetic pathway of Catharanthus roseus cells, and stimulates the accumulation of defense-related protein PR1 in the cells, showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway. Thus, our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.
Science in China Series C Life Sciences 05/2007; 50(2):234-41. · 1.61 Impact Factor
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ABSTRACT: Nitric oxide (NO) has emerged as a key signaling molecule in plant secondary metabolite biosynthesis recently. In order to investigate the molecular basis of NO signaling in elicitor-induced secondary metabolite biosynthesis of plant cells, we determined the contents of NO, salicylic acid (SA), jasmonic acid (JA), and puerarin in Pueraria thomsonii Benth. suspension cells treated with the elicitors prepared from cell walls of Penicillium citrinum. The results showed that the fungal elicitor induced NO burst, SA accumulation and puerarin production of P. thomsonii Benth. cells. The elicitor-induced SA accumulation and puerarin production was suppressed by nitric oxide specific scavenger cPITO, indicating that NO was essential for elicitor-induced SA and puerarin biosynthesis in P. thomsonii Benth. cells. In transgenic NahG P. thomsonii Benth. cells, the fungal elicitor also induced puerarin biosynthesis, NO burst, and JA accumulation, though the SA biosynthesis was impaired. The elicitor-induced JA accumulation in transgenic cells was blocked by cPITO, which suggested that JA acted downstream of NO and its biosynthesis was controlled by NO. External application of NO via its donor sodium nitroprusside (SNP) enhanced puerarin biosynthesis in transgenic NahG P. thomsonii Benth. cells, and the NO-triggered puerarin biosynthesis was suppressed by JA inhibitors IBU and NDGA, which indicated that NO induced puerarin production through a JA-dependent signal pathway in the transgenic cells. Exogenous application of SA suppressed the elicitor-induced JA biosynthesis and reversed the inhibition of IBU and NDGA on elicitor-induced puerarin accumulation in transgenic cells, which indicated that SA inhibited JA biosynthesis in the cells and that SA might be used as a substitute for JA to mediate the elicitor- and NO-induced puerarin biosynthesis. It was, therefore, concluded that NO might mediate the elicitor-induced puerarin biosynthesis through SA- and JA-dependent signal pathways in wildtype P. thomsonii Benth. cells and transgenic NahG cells respectively.
Science in China Series C Life Sciences 09/2006; 49(4):379-89. · 1.61 Impact Factor
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ABSTRACT: Nitric oxide and reactive oxygen species are two important signal molecules that play key roles in plant defense responses.
Nitric oxide generation and oxidative burst and accumulation of reactive oxygen species are the early reactions of Taxus chinensis suspension cells to fungal elicitor prepared from the cell walls of Penicillium citrinum. In order to investigate the relationship and/or interactions of nitric oxide and reactive oxygen species in the elicitor-induced
Taxol biosynthesis of T. chinensis suspension cells, we treated the cells with nitric oxide specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide
(cPITO), nitric oxide synthase inhibitor S,S′-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU), membrane NAD(P) H
oxidase inhibitor diphenylene iodonium (DPI), superoxide dismutases (SOD) and catalase. The results show that pretreatment
of T. chinensis cells with cPITO and DPI inhibited not only the elicitor-induced nitric oxide biosynthesis and oxidative burst, but also
the elicitor-induced Taxol production, suggesting that both nitric oxide and reactive oxygen species are involved in elicitor-induced
Taxol biosynthesis. Furthermore, pretreatment of the cells with cPITO and PBITU suppressed the elicitor-induced oxidative
burst, indicating that the oxidative burst might be dependent on NO. Application of nitric oxide via its donor sodium nitroprusside
(SNP) triggered Taxol biosynthesis of T. chinensis cells. The nitric oxide-induced Taxol production was suppressed by DPI, showing that the oxidative burst is involved in NO-triggered
Taxol biosynthesis. However, nitric oxide and the fungal elicitor induced Taxol biosynthesis even though the accumulation
of reactive oxygen species wass completely abolished in T. chinensis cells. Our data show that nitric oxide may mediate the elicitor-induced Taxol biosynthesis of T. chinensis suspension cells through both reactive oxygen species-dependent and-independent signal pathways. Moreover, the results of
our work show that the elicitor-and nitric oxide-induced Taxol biosynthesis is inhibited by catalase, indicating that H2O2 from the oxidative burst might be the signal molecule involved in induced Taxol production of T. chinensis cells.
Chinese Science Bulletin 07/2006; 51(16):1967-1975. · 1.32 Impact Factor
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ABSTRACT: Elicitor prepared from the cell walls of Penicillium citrinum induced multiple responses in Catharanthus roseus suspension cells, including rapid generation of nitric oxide (NO), sequentially followed by enhancement of catharanthine production by C. roseus cells. Elicitor-induced catharanthine biosynthesis was blocked by NO-specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and nitric oxide synthase (NOS) inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU). PBITU also strongly inhibited elicitor-induced NO generation by C. roseus suspension cells. The inhibiting effect of PBITU on elicitor-induced catharanthine production was reversed by external application of NO via the NO-donor sodium nitroprusside. The results strongly suggested that NO, generated by NOS or NOS-like enzymes in C. roseus suspension cells when treated with the fungal elicitor, was essential for triggering catharanthine synthesis.
Applied Microbiology and Biotechnology 05/2005; 67(1):40-4. · 3.42 Impact Factor
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ABSTRACT: Sodium nitroprusside (SNP) was used as the donor of nitric oxide (NO) to investigate its effect on catharanthine synthesis and the growth of Catharanthus roseus suspension cells. The results showed that SNP at high concentrations (10.0 and 20.0 mmol/L) stimulated catharanthine formation of C. roseus cells, but inhibited growth of the cells. Low concentrations of SNP (0.1 and 0.5 mmol/L) enhanced the growth of C. roseus cells, but had no effect on catharanthine synthesis. The maximum total catharanthine production was achieved by the addition of 0.5 and 10.0 mmol/L SNP to the cultures at day 0 and day 10, respectively, being about threefold of the control. NO-induced catharanthine production of C. roseus cells was strongly suppressed by jasmonic acid (JA) biosynthesis inhibitor ibuprofen (IBU) and nordihydroguaiaretic (NDGA). The result suggests that the stimulatory role of NO on catharanthine production is partially JA-dependent.
Biotechnology and Bioengineering 03/2005; 89(3):367-71. · 3.95 Impact Factor
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ABSTRACT: Elicitor, derived from the cell walls of Aspergillus niger, induced rapid generation of reactive oxygen intermediates (ROI), including superoxide anion (O2−) and hydrogen peroxide (H2O2), sequentially followed by phenylalanine ammonia-lyase (PAL) activation and catharanthine biosynthesis in Catharanthus roseus suspension cells. The elicitor-induced PAL activation and catharanthine biosynthesis were blocked by NAD(P)H oxidase inhibitor, diphenylene iodonium (DPI). O2− generated by the reaction of xanthine/xanthine oxidase (X/XO) triggered PAL activation and catharanthine biosynthesis of C. roseus cells in the absence of elicitor and reversed the inhibitory effect of DPI on elicitor-induced PAL activation and catharanthine biosynthesis. External application of H2O2 and catalase had no effect on PAL activity and catharanthine contents of C. roseus cells. The results demonstrated a causal relationship between elicitor-induced oxidative burst and PAL activation in C. roseus suspension cells and suggested a sequence of signaling events from ROI production to PAL activation and catharanthine synthesis. Within this sequence, O2− rather than H2O2 appeared to trigger the subsequent reactions.
Enzyme and Microbial Technology.
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ABSTRACT: Nitric oxide (NO) mediates a signal pathway that is involved in many defense responses of plants to biotic and abiotic stresses such as the hypersensitive cell death and the activation of defense-related genes. To determine the role of NO in secondary metabolite production of plant cells, sodium nitroprusside (SNP) is utilized as the donor of NO to investigate its effect on the production of terpenoid indole alkaloids of Catharanthus roseus cell suspension cultures. The results showed that the production of ajmalicine, catharanthine, and total alkaloids of C. roseus cells treated with 10.0 mmol/L SNP were 20.1, 24.2 and 51.3 mg/L, being 1.6-, 2.9- and 1.8-fold higher than those of untreated cells respectively. SNP-induced terpenoid indole alkaloid production of the cells was blocked by NO specific scavenger 2-4-carboxypheny-l-4,4,5,5-tetramethylimidazoline-1-oxyl-3- oxide (CPITO), which indicated that the effects of the application of SNP were caused by NO released from it rather than SNP itself. SNP treatment also induced the activation of protein kinases in C. roseus cell cultures. Pretreatment of the cells with protein kinase inhibitor K-252a suppressed SNP-induced terpenoid indole alkaloid production. The results strongly suggested that NO released from SNP triggered terpenoid indole alkaloid biosynthesis of C. roseus cells through a protein kinase-dependent signal pathway.
Enzyme and Microbial Technology.
