Elevated CO2 atmosphere enhances production of defense-related flavonoids in soybean elicited by NO and a fungal elicitor

Increased atmospheric pollutants including carbon dioxide (CO₂) and nitric oxide (NO) have a large impact on vegetation, with detrimental or beneficial influences on plant growth and metabolism. Here, we evaluated the effect of an elevated CO₂ atmosphere on the production of soybean defensive secondary chemicals induced by NO and a fungal elicitor. We hypothesized that an excess of carbon may alter the production of specific flavonoids that were previously shown to be induced by NO in soybean cotyledons. Pots containing soybean seeds (Glycine max [L.] Merr.) were submitted to 380 and 760 μmol mol^?1 of atmospheric CO₂ in open-top chambers. After nine days, plantlets grown under these conditions were assessed for biochemical and physiological parameters. Defense-related flavonoids were evaluated in detached cotyledon diffusates elicited with two different NO donors and with the β-glucan elicitor from Phytophthora sojae. A CO₂-enriched atmosphere stimulated initial growth, photosynthetic assimilation, and an altered C/N ratio in soybean plantlets resulting in allocation of precursors into different branches of the phenylpropanoid pathway in the cotyledons. Under elevated CO₂, the biotic elicitor caused accumulation of phytoalexins (glyceollins) as the natural end products of the phenylpropanoid pathway. In contrast, elevated CO₂ combined with NO resulted in an increase of intermediates and diverted end products (daidzein—127%, coumestrol—93%, genistein—93%, luteolin—89% and apigenin—238%) with a concomitant increase of 1.5–3.0 times in the activity of enzymes related to their biosynthetic routes. These observations point to changes in the pool of defense-related flavonoids that are related to increased carbon availability in soybeans. This may alter the responsiveness of soybean plants to pathogens when they are grown in CO₂ atmospheric concentrations close to those predicted for the upcoming several decades.     

Host-Pathogen Interactions : XXI. Extraction of a Heat-Labile Elicitor of Phytoalexin Accumulation from Frozen Soybean Stems

An extract of frozen and thawed soybean (Glycine max L. Merr. cv. Wayne) stems is active, in wounded soybean cotyledons, as a heat-labile elicitor of phytoalexins. The elicitor activity of the extract is destroyed by heating to 95°C for 10 minutes. The fraction that contains heat-labile elicitor activity releases heat-stable elicitor-active molecules from purified soybean cell walls. Heat-labile elicitor activity voids a Bio-Gel P-6 column and can be absorbed onto and eluted from a DEAE Sephadex ion exchange column. Using the cotyledon phytoalexin elicitor assay, maximum heatlabile elicitor activity was obtained when soybean stems were extracted with acetate buffer at pH 6.0. Addition of 1 millimolar CaCl₂ increased apparent heat-labile elicitor activity. The heat-labile elicitor stimulated maximum phytoalexin accumulation when applied to cotyledons immediately after the cotyledons were cut. Partially purified stem extracts lost heat-labile elicitor activity during storage for several days at 3°C. The possible role of a heat-labile elicitor in stimulation of phytoalexin accumulation by both abiotic and biotic elicitors is discussed.     

Host-Pathogen Interactions: IX. Quantitative Assays of Elicitor Activity and Characterization of the Elicitor Present in the Extracellular Medium of Cultures of Phytophthora megasperma var. sojae

Resistance of soybean (Glycine max L.) seedlings to Phytophthora megasperma var. sojae (Pms) is in part due to the accumulation in infected tissue of a compound which is toxic to Pms. The accumulation of this compound, a phytoalexin called glyceollin, is triggered by infection, but it can also be triggered by molecules, “elicitors,” present in cultures of Pms. The ability of the Pms elicitor to stimulate phytoalexin accumulation in soybean tissues has been used as the basis for biological assays of elicitor activity. Two bioassays were developed and characterized in this study of the Pms elicitor. These bioassays use the cotyledons and the hypocotyls of soybean seedlings. The cotyledon assay was used to characterize the extracellular Pms elicitor. This elicitor was isolated from Pms cultures and purified by ion exchange and molecular sieving chromatography. The extracellular Pms elicitor was determined to be a predominantly 3-linked glucan, which is similar in composition and structure to a polysaccharide component of Pms mycelial walls.     

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 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.     

Rapid Accumulation of Anionic Peroxidases and Phenolic Polymers in Soybean Cotyledon Tissues following Treatment with Phytophthora megasperma f. sp. Glycinea Wall Glucan

Phytophthora megasperma Drechs. f. sp. glycinea Kuan & Erwin (PMG) cell wall glucan has been extensively characterized as an elicitor of the pterocarpan phytoalexins, the glyceollins in soybean (Glycine max L.). Just recently, this glucan was shown to be a potent elicitor of conjugates of the isoflavones, daidzein and genistein as well. Here we report that PMG wall glucan also induces a rapid and massive accumulation of phenolic polymers in soybean cotyledon cells proximal to the point of elicitor application. Deposition of phenolic polymers is over then times that in wounded controls within just 4 hours of elicitor treatment and reaches a maximum by 24 hours. In the same tissues, isoflavone conjugates begin to accumulate at 8 hours and glyceollin at 12 hours. By 24 hours, the total deposition of wall bound phenolics in elicitor-treated tissues is several times greater than the peak glyceollin and isoflavone responses combined. Histochemical stains and quantitation of phenolic residues released after saponification and nitrobenzene or copper oxide oxidation suggest that the covalently linked phenolics include both lignin- and suberin-like polymers as well as simple esterified coumaric and ferulic acid monomers. Accumulations of phenolic polymers are accompanied by equally rapid and massive increases in activity of a specific group of anionic peroxidases. Although increases in peroxidase activity are not strictly limited to cells immediately adjacent to the area of elicitor treatment, the deposition of phenolic polymers is significantly less extensive in distal cells.     

Phytoalexin Elicitor Activity of Carbohydrates from Phytophthora megasperma f.sp. glycinea and Other Sources

Three unique classes of carbohydrates were isolated from the hyphal cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) and compared with other substances for their activity as elicitors of the phytoalexin glyceollin in soybean tissues. Glucomannans extracted from cell walls with soybean β-1,3-endoglucanase were purified and proved to be the most active elicitors yet reported. They were approximately 10 times more active in soybean cotyledons than the heterogeneous β-glucan elicitor fraction extracted from Pmg walls. In addition, the glucomannan fraction gave race-specific elicitor activity in soybean hypocotyls. Pronase was found to be a suitable reagent for the mild extraction of glycopeptides from Pmg cell walls. All of the carbohydrates isolated from Pmg cell walls possessed significant elicitor activity, but other glucans, a glucomannan and mannan from other sources, were much less active. Chitin and chitosan, reported to function as elicitors in other plants, had low activity in soybean cotyledons. Arachidonic acid was inactive, despite its previously observed elicitor activity in potato tubers. The results indicated that, for Pmg, the carbohydrate elicitor most probably involved in the initiation of phytoalexinmediated defense during fungus infection of soybean plants is the glucomannan fraction liberated by endoglucanase.     

Release of a Soluble Phytoalexin Elicitor from Mycelial Walls of Phytophthora megasperma var. sojae by Soybean Tissues

A soluble elicitor of glyceollin accumulation was released from insoluble mycelial walls of Phytophthora megasperma var. sojae after incubation with soybean cotyledon tissue for as little as 2 minutes. Various enzymic and chemical treatments of the released elicitor indicated that the activity resided in a carbohydrate moiety, and gel filtration disclosed the presence of at least two active molecular species. Cell-free extracts from soybean cotyledons or hypocotyls also released soluble elicitors from fungal cell walls that were similar to those released by living cotyledon tissue. These results may suggest that contact of fungal pathogens with host tissues is required to release fungal wall elicitors which then initiate phytoalexin accumulation in the plant.     

Signaling Effects of Nitric Oxide, Salicylic Acid, and Reactive Oxygen Species on Isoeuphpekinensin Accumulation in Euphorbia pekinensis Suspension Cells Induced by an Endophytic Fungal Elicitor

Nitric oxide (NO), salicylic acid (SA), and reactive oxygen species (ROS) are important signal molecules that mediate plant resistance reactions and play important roles in secondary metabolism. To research the signal transduction pathway of the endophytic fungal elicitor from Fusarium sp. E5 promoting secondary metabolism in Euphorbia pekinensis suspension cells, the changes in NO, SA, ROS, and isoeuphpekinensin contents in the cells were investigated after elicitor addition to the cell suspension culture. The elicitor did not change H₂O₂ or O₂ ^? contents notably, whereas NO and SA contents were enhanced. Both the NO donator sodium nitroprusside (SNP) and SA enhanced isoeuphpekinensin content in the absence of the fungal elicitor, whereas the NO scavenger cPTIO and SA biosynthesis inhibitor cinnamic acid (CA) inhibited isoeuphpekinensin accumulation in the presence of the elicitor. In addition, cPTIO inhibited SA production induced by the fungal elicitor. CA did not inhibit NO production, but it significantly inhibited isoeuphpekinensin accumulation. The results demonstrated that in Euphorbia pekinensis suspension cells the endophytic fungal elicitor induced increased NO content and SA production, which promoted isoeuphpekinensin accumulation. ROS are clearly not involved in the endophytic fungus–host interaction signaling pathway.     

Sulfhydryl-reagent alteration of soybean resistance to the cabbage looper, Trichoplusia ni

The hypothesis that the sulfhydryl reagent, N-ethylmaleimide, would function as an elicitor of alterable resistance in soybean (Glycine max) plants to Trichoplusia ni herbivory was tested experimentally under greenhouse conditions. This elicitory chemical, which allows receptor thiols to add across its carbon-carbon double bond, altered the resistance in one or more leaves of plants at one or more intervals after treatment; and thus yielded results supporting the hypothesis. Leaf dipping and soil application were both effective methods of treatment. Results support the interpretation that an elicitor may function in intact plants by altering the integrity of sulfhydryl groups in receptor macromolecules which are also involved in signaling a change in the plant's biosynthesis of characteristic defensive compounds such as phenylpropanoids including antifeedant and antibiotic flavonoids. Induced feeding non-preference by T. ni was highly correlated positively with the amount of glyceollins in the leaf.     

Role of nitric oxide in UV-B-induced activation of PAL and stimulation of flavonoid biosynthesis in Ginkgo biloba callus

The role of nitric oxide (NO) in UV-B-induced secondary metabolite accumulation in Ginkgo biloba callus was investigated. Overall, UV-B irradiation induced multiple biological responses in callus of G. biloba, including increased both NO production and nitric oxide synthase (NOS) activity, and subsequent activation of phenylalanine ammonium lyase (PAL) and synthesis of flavonoids. Application of NO via the donor sodium nitroprusside (SNP) enhanced UV-B-induced PAL activity and increased accumulation of flavonoids in G. biloba callus. Both, the NOS inhibitor l-NAME (N (G)-nitro-l-arginine methyl ester) and the NO scavenger c-PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide) reduced the production of NO. Moreover, UV-B-induced increase of PAL activity and flavonoid accumulation were suppressed by l-NAME and c-PTIO. These findings suggested a causal relationship between NO release and both PAL activity and flavonoid accumulation under UV-B irradiation. In addition, it also indicated that NO, produced via NOS-like activity in ginkgo callus subjected to UV-B irradiation, might act as an essential signaling molecule for triggering the activation of PAL and synthesis of flavonoids. Additionally, a guanylyl cyclase inhibitor 6-anilino-5,8-quinolinequinone (LY-83583) prevented both UV-B- and SNP-induced enhancement of PAL activation and flavonoid biosynthesis thus suggesting that the NO function was mediated by cyclic guanosine 5’-monophosphate. However, these effects of c-PTIO, l-NAME, and LY-83583 were partial, thus suggesting that there were NO-independent pathways in UV-B signaling networks. Gangping Hao and Xihua Du are contributed equally to this article.     

Role of nitric oxide in abscisic acid-induced subcellular antioxidant defense of maize leaves

The sources of nitric oxide (NO) production in response to abscisic acid (ABA) and the role of NO in ABA-induced hydrogen peroxide (H(2)O(2)) accumulation and subcellular antioxidant defense in leaves of maize (Zea mays L.) plants were investigated. ABA induced increases in generation of NO and activity of nitric oxide synthase (NOS) in maize leaves. Such increases were blocked by pretreatment with each of the two NOS inhibitors. Pretreatments with a NO scavenger or NR inhibitors inhibited ABA-induced increase in production of NO, but did not affect the ABA-induced increases in activity of NOS, indicating that ABA-induced NO production originated from sources of NOS and NR. ABA- and H(2)O(2)-induced increases in expression of the antioxidant genes superoxide dismutase 4 (SOD4), cytosolic ascorbate peroxidase (cAPX), and glutathione reductase 1 (GR1) and the activities of the chloroplastic and cytosolic antioxidant enzymes were arrested by pretreatments with the NO scavenger, inhibitors of NOS and NR, indicating that NO is involved in the ABA- and H(2)O(2)-induced subcellular antioxidant defense reactions. On the other hand, NO donor sodium nitroprusside (SNP) reduced accumulation of H(2)O(2) induced by ABA, and c-PTIO reversed the effect of SNP in decreasing the accumulation of H(2)O(2). SNP induced increases in activities of subcellular antioxidant enzymes, and the increases were substantially prevented from occurring by the pretreatment with c-PTIO. These results suggest that ABA induces production of H(2)O(2) and NO, which can up-regulate activities of the subcellular antioxidant enzymes, to prevent overproduction of H(2)O(2) in maize plants. There is a negative feedback loop between NO and H(2)O(2) in ABA signal transduction in maize plants.     

Nitric oxide mediates either proliferation or cell death in cardiomyocytes. Involvement of polyamines

Summary Nitric oxide (NO) is a molecule involved in several signal transduction pathways leading either to proliferation or to cell death. Induction of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, represents an early event preceding DNA synthesis. In some cell types increased ODC activity seems to be involved in cytotoxic response. We investigated the role of NO and ODC induction on the events linked to cell proliferation or to cell death in cultured chick embryo cardiomyocytes. Exposure of cardiomyocytes to tumor necrosis factor (TNF) and lipopolysaccharide (LPS) caused NO synthase (NOS) and ODC induction as well as increased incorporation of [³H]-thymidine. This last effect was blocked by a NOS inhibitor and was strongly reduced by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC. Sodium nitroprusside (SNP), an exogenous NO donor, inhibited the increases of NOS and ODC activities and abolished the mitogenic effect of TNF and LPS. Moreover, SNP alone caused cell death in a dose dependent manner. The cytotoxicity of SNP was not affected by DFMO while it was prevented by antioxidants. The results suggest that different pathways would mediate the response of cardiomyocytes to NO: they can lead either to ODC induction and DNA synthesis when NO is formed through NOS induction or to growth inhibition and cell death, when NO is supplied as NO donor. Increased polyamine biosynthesis would mediate the proliferative response of NO, while the cytotoxicity of exogenous NO seems to involve some oxidative reactions and to depend on the balance between NO availability and cellular redox mechanisms.     

Fungal endophytes-induced abscisic acid is required for flavonoid accumulation in suspension cells of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Treatment of suspension cells of Ginkgo biloba with fungal endophytes resulted in accumulation of flavonoids, increased abscisic acid (ABA) production and activation of phenylalanine ammonia-lyase (PAL). Fluridone, an inhibitor of ABA biosynthesis, was effective in inhibiting fungal endophytes-induced ABA biosynthesis, increase of PAL activity and flavonoids accumulation. Moreover, exogenous application of ABA enhanced PAL activity and increased accumulation of flavonoids in G. biloba cells with or without fungal endophytes elicitor. These finding suggest a causal relationship between ABA release and both PAL activity and flavonoid accumulation under fungal endophytes treatment and that ABA is involved in fungal endophytes-induced flavonoids accumulation in this plant.     

Role of nitric oxide dependence on nitric oxide synthase-like activity in the water stress signaling of maize seedling

Nitric oxide (NO) has been known as an important signal in plant antioxidative defense but its production and roles in water stress are less known. The present study investigated whether NO dependence on a NO synthase-lika (NOS) activity is involved in the signaling of drought-induced protective responses in maize seedlings. NOS activity, rate of NO release and drought responses were analyzed when NO donor sodium nitroprusside (SNP), NO scavenger c-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramathylimidazoline-1-oxyl-3-oxide) and NOS inhibitor L-NAME (NG-nitro-L-arginine methyl ester) were applied to both detached maize leaves and whole plants. Both NOS activity and the rate of NO release increased substantially under dehydration stress. The high NOS activity induced by c-PTIO as NO scavenger and NO accumulation Inhibited by NOS inhibitor L-NAME In dehydration-treated maize seedlings Indicated that most NO production under water deficit stress may be generated from NOS-like activity. After dehydration stress for 3 h, detached maize leaves pretreated with NO donor SNP maintained more water content than that of control leaves pretreated with water. This result was consistent with the decrease in the transpiration rate of SNP-treated leaves subjected to drought treatment for 3 h. Membrane permeability, a cell injury index, was lower in SNP-trested maize leaves under dehydration stress for 4 h when compared with the control leaves. Also, superoxide dismutsse (SOD) activity of SNP combined drought treatment maize leaves was higher than that of drought treatment alone, indicating that exogenous NO treatment alleviated the water loss and oxidative damage of maize leaves under water deficit stress. When c-PTIO as a specific NO scavenger was applied, the effects of applied SNP were overridden. Treatment with L-NAME on leaves also led to higher membrane permeability, higher transpiration rate and lower SOD activities than those of control leaves, indicating that NOS-like activity was involved in the antioxidative defense under water stress. These results suggested that NO dependence on NOS-like activity serves as a signaling component in the induction of protective responses and is associated with drought tolerance in maize seedlings.     

Quantification of glyceollins in non-elicited seedlings of Glycine max by gas chromatography-mass spectrometry

A sensitive gas chromatography-mass spectrometry method is described for detection and quantification of small amounts of phytoalexins, especially glyceollins. This method allowed identification of canescacarpin (glyceollin V) besides appreciable amounts of glyceollin isomers I–III and glyceofuran in unchallenged parts of soybean seedlings. Based on this quantification method the glyceollin content of soybean roots, either untreated, wounded or incubated with a glucan elicitor from Phytophthora sojae, was compared. The proportions of glyceollin isomers in the mixture of compounds were determined in different soybean tissues. Single-ion monitoring revealed the presence of four additional glyceollin isomers with benzofuranoid structure.     

Structure-activity relationships of oligo-beta-glucoside elicitors of phytoalexin accumulation in soybean.

The abilities of a family of chemically synthesized oligo-beta-glucosides, ranging in size from hexamer to decamer, to induce phytoalexin accumulation in soybean cotyledons were investigated to determine which structural elements of the oligoglucosides are important for their biological activity. The results of the biological assays established that the following structural motif is necessary for the oligo-beta-glucosides to have high elicitor activity: [formula; see text] The branched trisaccharide at the nonreducing end of the oligoglucosides was found to be essential for maximum elicitor activity. Substitution of either the nonreducing terminal backbone glucosyl residue or the side-chain glucosyl residue closest to the nonreducing end with glucosaminyl or N-acetylglucosaminyl residues reduced the elicitor activity of the oligoglucosides between 10-fold and 10,000-fold. Elicitor activity was also reduced 1000-fold if the two side-chain glucosyl residues were attached to adjacent backbone glucosyl residues rather than to glucosyl residues separated by an unbranched residue. In contrast, modifications of the reducing terminal glucosyl residue of an elicitor-active hepta-beta-glucoside by conjugation with tyramine and subsequent iodination had no significant effect on the elicitor activity of the hepta-beta-glucoside. These results demonstrate that oligo-beta-glucosides must have a specific structure to trigger the signal transduction pathway, which ultimately leads to the de novo synthesis of phytoalexins in soybean.     

Protective effect of exogenously applied nitric oxide on aluminum-induced oxidative stress in soybean plants

Aluminum (Al) toxicity promotes oxidative damage in plants, while nitric oxide (NO) may exert a beneficial effect on Al toxicity condition in soybean. Pretreatment with NO donor sodium nitroprusside (SNP) before soybean exposure to Al significantly reduced Al accumulation and MDA induction in the root apex. Pretreatment with SNP also increased the relative root elongation, chlorophyll content, and activity of the protective enzyme peroxidase compared to Al treatment alone. These results show the effect of exogenously applied NO as a protector against oxidative stress induced by Al. Moreover, the ameliorating effect can be reversed by the addition of NO scavenger 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) in the presence of Al.     

Reactive oxygen species, nitric oxide, and their interactions play different roles in Cupressus lusitanica cell death and phytoalexin biosynthesis

Beta-thujaplicin Is a natural troponoid with strong antifungal, antiviral, and anticancer activities. Beta-thujaplicin production in yeast elicitor-treated Cupressus lusitanica cell culture and its relationships with reactive oxygen species (ROS) and nitric oxide (NO) production and hypersensitive cell death were investigated. Superoxide anion radical (O2*-) induced cell death and inhibited beta-thujaplicin accumulation, whereas hydrogen peroxide (H2O2) induced beta-thujaplicin accumulation but did not significantly affect cell death. Both elicitor and O2*- induced programmed cell death, which can be blocked by protease inhibitors, protein kinase inhibitors, and Ca2+ chelators. Elicitor-induced NO generation was nitric oxide synthase (NOS)-dependent. Inhibition of NO generation by NOS inhibitors and NO scavenger partly blocked the elicitor-induced beta-thujaplicin accumulation and cell death, and NO donors strongly induced cell death. Interaction among NO, H2O2, and O2*- shows that NO production and H2O2 production are interdependent, but NO and O2*- accumulation were negatively related because of coconsumption of NO and O2*-. NO- and O2*- -induced cell death required each other, and both were required for elicitor-induced cell death. A direct interaction between NO and O2*- was implicated in the production of a potent oxidant peroxynitrite, which might mediate the elicitor-induced cell death.