UV-B irradiation alleviates the deterioration of cold-stored mangoes by enhancing endogenous nitric oxide levels.

Effects of UV-B radiation on chilling injury, ripening and endogenous nitric oxide (NO) levels in mango fruit were evaluated. Chilling injury index, ion leakage, and malondialdehyde (MDA) content of the fruit pretreated with 5kJm(-2) UV-B for 4h were significantly lower than those of the control during fruit ripening at ambient temperature following cold storage at 6°C for 10days. Fruit firmness of the mangoes irradiated with UV-B was significantly higher than the control during the ripening period. Endogenous NO levels of the UV-B-irradiated fruit were rapidly increased after UV-B treatment. Pre-treatment of mangoes with the NO specific scavenger, not only abolished UV-B-triggered NO accumulation, but also suppressed the UV-B-reduced chilling injury, oxidative damage, and ripening delay of the fruit. Together, the results suggest that UV-B treatment may enhance chilling tolerance and delay fruit ripening of mangoes by triggering endogenous NO generation in the fruit.

Nitric oxide fumigation stimulates flavonoid and phenolic accumulation and enhances antioxidant activity of mushroom.

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.

Ultraviolet-B-induced flavonoid accumulation in Betula pendula leaves is dependent upon nitrate reductase-mediated nitric oxide signaling.

Nitric oxide (NO) is an important signaling molecule involved in many physiological processes in plants. Nitric oxide generation and flavonoid accumulation are two early reactions of plants to ultraviolet-B (UV-B) irradiation. However, the source of UV-B-triggered NO generation and the role of NO in UV-B-induced flavonoid accumulation are not fully understood. In order to evaluate the origin of UV-B-triggered NO generation, we examined the responses of nitrate reductase (NR) activity and the expression levels of NIA1 and NIA2 genes in leaves of Betula pendula Roth (silver birch) seedlings to UV-B irradiation. The data show that UV-B irradiation stimulates NR activity and induces up-regulation of NIA1 but does not affect NIA2 expression during UV-B-triggered NO generation. Pretreatment of the leaves with NR inhibitors tungstate (TUN) and glutamine (Gln) abolishes not only UV-B-triggered NR activities but also UV-B-induced NO generation. Furthermore, application of TUN and Gln suppresses UV-B-induced flavonoid production in the leaves and the suppression of NR inhibitors on UV-B-induced flavonoid production can be reversed by NO via its donor sodium nitroprusside. Together, the data indicate that NIA1 in the leaves of silver birch seedlings is sensitive to UV-B and the UV-B-induced up-regulation of NIA1 may lead to enhancement of NR activity. Furthermore, our results demonstrate that NR is involved in UV-B-triggered NO generation and NR-mediated NO generation is essential for UV-B-induced flavonoid accumulation in silver birch leaves.

Abscisic acid plays critical role in ozone-induced taxol production of Taxus chinensis suspension cell cultures.

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.

Enhancing hypericin production of Hypericum perforatum cell suspension culture by ozone exposure.

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.

Nitrate reductase-mediated nitric oxide generation is essential for fungal elicitor-induced camptothecin accumulation of Camptotheca acuminata suspension cell cultures.

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.

Complementary action of jasmonic acid on salicylic acid in mediating fungal elicitor-induced flavonol glycoside accumulation of Ginkgo biloba cells.

The antagonistic action between jasmonic acid (JA) and salicylic acid (SA) in plant defence responses has been well documented. However, their relationship in secondary metabolite production is largely unknown. Here, we report that PB90, a protein elicitor from Phytophthora boehmeriae, triggers JA generation, SA accumulation and flavonol glycoside production of Ginkgo biloba cells. JA inhibitors suppress not only PB90-triggered JA generation, but also the elicitor-induced flavonol glycoside production. However, the elicitor can still enhance flavonol glycoside production even though the JA generation is totally inhibited. Over-expression of SA hydrolase gene NahG not only abolishes SA accumulation, but also suppresses the elicitor-induced flavonol glycoside production when JA signalling is inhibited. Interestingly, expression of NahG does not inhibit the elicitor-induced flavonol glycoside accumulation in the absence of JA inhibitors. Moreover, JA levels are significantly enhanced when SA accumulation is impaired in the transgenic cells. Together, the data suggest that both JA and SA are involved in PB90-induced flavonol glycoside production. Furthermore, we demonstrate that JA signalling might be enhanced to substitute for SA to mediate the elicitor-induced flavonol glycoside accumulation when SA signalling is impaired, which reveals an unusual complementary relationship between JA and SA in mediating plant secondary metabolite production.

Synergistic action between jasmonic acid and nitric oxide in inducing matrine accumulation of Sophora flavescens suspension cells.

Secondary metabolites not only play important ecological roles in plants but also are important pharmaceutical and source compounds for derivative synthesis. Production of plant secondary metabolites is believed to be controlled by the endogenous signal network of plants. However, the molecular basis is still largely unknown. Here we show that matrine production of Sophora flavescens Ait. cells treated with low levels of jasmonic acid (JA) and nitric oxide (NO) is significantly increased although treatment with low concentrations of JA or NO alone has no effects on matrine production, showing that JA and NO may act synergistically in triggering matrine production. Moreover, treatment with NO triggers lipoxygenase (LOX) activity and enhances JA levels of the cells, showing that NO may activate the endogenous JA biosynthesis of S. flavescens cells. External application of JA induces nitric oxide synthase-like activities and stimulates NO generation of S. flavescens cells, which suggests that JA may trigger NO generation of the cells. Thus, the results reveal a mutually amplifying reaction between JA and NO in S. flavescens cells. Furthermore, JA and NO inhibitors suppress not only the mutually amplifying reaction between JA and NO but also the synergistic effects of NO and JA on matrine production. Therefore, the data demonstrate that the synergistic action of JA and NO in inducing matrine production might be due to the mutually amplifying reaction between JA and NO in the cells.

Signal interaction between nitric oxide and hydrogen peroxide in heat shock-induced hypericin production of Hypericum perforatum suspension cells.

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.

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 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.

Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells.

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.

Nitric oxide mediates the fungal elicitor-induced puerarin biosynthesis in Pueraria thomsonii Benth. suspension cells through a salicylic acid (SA)-dependent and a jasmonic acid (JA)-dependent signal pathway.

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.

Nitric oxide mediates the fungal elicitor-induced hypericin production of Hypericum perforatum cell suspension cultures through a jasmonic-acid-dependent signal pathway.

Fungal elicitor prepared from the cell walls of Aspergillum niger induces multiple responses of Hypericum perforatum cells, including nitric oxide (NO) generation, jasmonic acid (JA) biosynthesis, and hypericin production. To determine the role of NO and JA in elicitor-induced hypericin production, we study the effects of NO scavenger 2- to 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, and inhibitors of the octadecanoid pathway on elicitor-induced NO generation, JA biosynthesis, and hypericin production. Pretreatment of the cells with cPITO and JA biosynthesis inhibitors suppresses not only the elicitor-induced NO generation and JA accumulation but also the elicitor-induced hypericin production, which suggests that both NO and JA are involved in elicitor-induced hypericin biosynthesis. S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea and cPITO inhibit both elicitor-induced NO generation and JA biosynthesis, while JA biosynthesis inhibitors do not affect the elicitor-induced NO generation, indicating that JA acts downstream of NO generation and that its biosynthesis is regulated by NO. External application of NO via its donor sodium nitroprusside induces hypericin production in the absence of fungal elicitor. Sodium-nitroprusside-induced hypericin production is blocked by JA biosynthesis inhibitors, showing that JA biosynthesis is essential for NO-induced hypericin production. The results demonstrate a causal relationship between elicitor-induced NO generation, JA biosynthesis, and hypericin production in H. perforatum cells and indicate a sequence of signaling events from NO to hypericin production, within which NO mediates the elicitor-induced hypericin biosynthesis at least partially via a JA-dependent signaling pathway.

[Enhancement of hypericin production and cell growth of Hypericum perforatum L. suspension cultures by nitric oxide].

Nitric oxide has emerged as a key signaling molecule in plants recently. The role of nitric oxide in elicitor-induced defense responses of plants has been extensively investigated. In this work, sodium nitroprusside was utilized as the donor of nitric oxide to investigate the effects of exogenous nitric oxide on hypericin production and cell growth of suspension cell cultures of Hypericum perforatum L.. Compared with the untreated Hypericum perforatum L. suspension cells, external application of 0.5 and 15.0 mmol/L sodium nitroprusside induced 1.4 and 0.5-fold dry cell weight, and 0.9 and 2.1-fold hypericin content respectively. The results showed that low concentration of sodium nitroprusside promoted the growth of Hypericum perforatum L. suspension cells, while high concentration of sodium nitroprusside enhanced hypericin biosynthesis in Hypericum perforatum L. suspension cells. The maximum hypericin production was achieved by adding 0.5 mmol/L and 15.0 mmol/L sodium nitroprusside to the culture at day 0 and day 14 respectively, increasing the total hypericin yield by nearly 3.2-fold. The effects of sodium nitroprusside on hypericin content and growth of Hypericum perforatum L. suspension cells were abolished by nitric oxide specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, which indicated that the effects of the application of sodium nitroprusside were caused by nitric oxide released from sodium nitroprusside rather than sodium nitroprusside itself. The results also showed that 15.0 mmol/L sodium nitroprusside stimulated the activities of phenylalanine ammonia-lyase (PAL), one of the key enzymes of phenylpropanoid pathway, in suspension cells of Hypericum perforatum L., which suggested that the synthetic pathway of hypericin might be activated by NO through triggering the defense responses of Hypericum perforatum L. suspension cells.

Effect of nitric oxide on catharanthine production and growth of Catharanthus roseus suspension cells.

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.

Elicitor-induced nitric oxide burst is essential for triggering catharanthine synthesis in Catharanthus roseus suspension cells.

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.