Protective Effect of Arachidonic Acid during Viral Infection: Synthesis of New Proteins by in vitroPotato Plants

The mechanisms of the protective, immunostimulating effects of arachidonic acid (AA) were studied, and its efficiency in the induction of defense reactions in the moderately virus-resistant potato cultivar Nevskii (Solanum tuberosumL.) was determined. Virus-free in vitropotato plants treated with AA and inoculated with phytopathogenic viruses were used as a model. The data on the X virus accumulation obtained by the enzyme-linked immunosorbent assay confirmed the immunizing effect of AA; the optimum concentration of the compound was 10^–8M. The antiviral effect of AA was maintained in infected in vitropotato plants for at least two or three weeks. The electrophoretic analysis of leaf proteins revealed a 33-kD polypeptide induced by the potato virus Y. Two weeks after inoculation with virus X, a 40-kD protein was identified in potato plants pretreated with AA. In addition, the relative content of the two groups of proteins consisting of two or three components with mol wts about 50 kD and above70 kD changed both upon viral infection and pretreatment with AA. Only small changes in the isozyme patterns of peroxidase in potato plants were observed during the development of systemic acquired resistance; they were manifested in some treatments in the band intensities. The existence of the alternative pathways of systemic acquired resistance in potato plants specifically activated by viral infection and AA was suggested.     

The Effect of Arachidonic Acid and Viral Infection on the Phytohemagglutinin Activity during the Development of Tobacco Acquired Resistance

The effects of arachidonic acid (AA) on the development of viral infection and the activity of phytohemagglutinins in Nicotiana tabacum L. plants were studied. Cv. Samsun NN was used, which displayed a genotypically determined hypersensitive response to tobacco mosaic virus (TMV) infection. When tobacco leaf disks were treated with 10^–9 to –10^–7 M AA, viral reproduction was suppressed by 90–100%. The AA concentration of 10^–8 M was optimal for the improvement of plant virus resistance. Tobacco leaves maintained virus resistance for at least two weeks. Both AA treatment and TMV inoculation were accompanied by an enhanced lectin activity, which may indicate the involvement of lectins in the development of plant defense responses. Lectin accumulation was observed in the intact plants developing systemic resistance and in the detached leaves characterized by local resistance.     

Influence of arachidonic acid and phytoviruses on the hormonal system of potato plants grown in vitro

Changes in the phytohormonal system during the development of antivirus resistance was investigated. This resistance was induced by arachidonic acid (AA) in potato plants (Solanum tuberosum L.) grown in vitro. The plants in tubes were treated with AA 7 days before infection with tobacco mosaic virus or potato X virus. These plants were used for ABA, IAA, and cytokinin quantification. As a rule, phytoviruses increased ABA and cytokinin levels and decreased that of IAA in both immunized and unimmunized plants. AA significantly decreased the phytovirus content in potato plants at least during 3 weeks. In general, AA immunization acted as a limiting factor for increasing the ABA and cytokinin concentrations during virus pathogenesis in experimental plants. However, no regularity in the changes of the IAA concentration was noted. The data obtained showed that AA-induced changes in the hormonal system could be an important factor in the mechanism of plant resistance against phytopatogenic viruses.     

Abscisic Acid Suppresses the Hypersensitive Necrosis of Potato Tubers and Reverses the Arachidonic Acid-Induced Inhibition of Plasma Membrane-Bound ATPase

Abscisic acid (ABA) fully suppressed the development of the hypersensitive necrosis of potato tuber slices to crude elicitor from Phytophthora infestans and arachidonic acid (AA) if supplied before or up to 6 to 8 h after elicitor treatments. Later application of ABA only reduced the necrotic response. Added to highly purified plasma membrane preparation from tuber tissue, ABA eliminated the AA-induced inhibition of the plasma membrane-bound (K⁺+ Na⁺+ Mg²⁺) ATPase. It is suggested that ABA counteracts AA in the membrane.     

Stimulation of Cellular Mechanisms of Potato Antivirus Resistance by the Action of a Preparation Based on <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Bacteria

A comparative study of the antiviral activity of a new biopreparation based on bacteria Bacillus subtilis (strain 47) and commercial biopesticides (beta-protectin, phyto-protectin, frutin) in potato plants of Belarusian selection (Lileya and Scarb) was carried out in vivo and ex vitro. Pretreatment of plants with B. subtilis biopreparation and biopesticides prevents infection by potato X-viruses. The antiviral efficacy of B. subtilis does not depend on the conditions of plant growth and is more effective than biopesticides. Increased potato plant resistance to viral infection was accompanied by an effect leading to an increase in the mass of minitubers and the dry matter content in them. Treatment of potato plants with B. subtilis did not affect the molecular heterogeneity of peroxidase and superoxide dismutase, but it changed the relative activity of their isoforms. Treatment with a bacterial preparation increased the activity of superoxide dismutase when it was applied both to intact plants and those preinfected with virus. The results indicate that pretreatment of potato plants with the B. subtilis drug prevents virus infection, inducing the antiviral resistance of the potato, and is accompanied by a change in the activity of redox enzymes.     

Effects of salicylic and jasmonic acids on the components of pro/antioxidant system in potato plants infected with late blight

The effects of salicylic acid (SA) and jasmonic acid (JA) on plant defense responses were studied with aseptic potato (Solanum tuberosum L.) plantlets infected with Phytophthora infestans (Mont.) de Bary. Plant treatment with 10⁻⁶ M SA or 10⁻⁷ M JA induced plant resistance; the mixture of these acids was most efficient. After treatment with these compounds, phenolic compounds were accumulated and peroxidase was activated in the sites of pathogen localization, and this might be the reason of resistance enhancement. In addition, more H₂O₂ was accumulated in infected plants treated with JA or its mixture with SA but not in plants treated with SA alone. It might occur because of observed inhibition of catalase and activation of isoperoxidase with the isoelectric point (pI) of ∼9.3, which manifests an affinity for the pathogen cell wall. The data obtained allow us to recommend the application of these compounds for potato plant protection against late blight.     

Effect of aristolochic acid on intracellular calcium concentration and its links with apoptosis in renal tubular cells

Aristolochic acid (AA) has been demonstrated to play a causal role in Chinese herbs nephropathy. However, the detailed mechanism for AA to induce apoptosis of renal tubular cells remains obscure. In this study, we show that AA evokes a rapid rise in the intracellular Ca²⁺ concentration of renal tubular cells through release of intracellular endoplasmic reticulum Ca²⁺ stores and influx of extracellular Ca²⁺, which in turn causes endoplasmic reticulum stress and mitochondria stress, resulting in activation of caspases and finally apoptosis. Ca²⁺ antagonists, including calbindin-D_28k (an intracellular Ca²⁺ buffering protein) and BAPTA-AM (a cell-permeable Ca²⁺ chelator), are capable of ameliorating endoplasmic reticulum stress and mitochondria stress, and thereby enhance the resistance of the cells to AA. Moreover, we show that overexpression of the anti-apoptotic protein Bcl-2 in combination with BAPTA-AM treatment can provide renal tubular cells with almost full protection against AA-induced cytotoxicity. In conclusion, our results demonstrate an impact of AA to intracellular Ca²⁺ concentration and its link with AA-induced cytotoxicity. Yi-Hong Hsin and Chi-Hung Cheng are equally contributed to this work.     

Glioblastoma cell death induced by asiatic acid

Asiatic acid (AA), a triterpene, is known to be cytotoxic to several tumor cell lines. AA induces dose- and time-dependent cell death in U-87 MG human glioblastoma. This cell death occurs via both apoptosis and necrosis. The effect of AA may be cell type-specific as AA-induced cell death was mainly apoptotic in colon cancer RKO cells. AA-induced glioblastoma cell death is associated with decreased mitochondrial membrane potential, activation of caspase-9 and -3, and increased intracellular free Ca²⁺. Although treatment of glioblastoma cells with the caspase inhibitor zVAD-fmk completely abolished AA-induced caspase activation, it did not significantly block AA-induced cell death. AA-induced cell death was significantly prevented by an intracellular Ca²⁺ inhibitor, BAPTA/AM. Taken together, these results indicate that AA induces cell death by both apoptosis and necrosis, with Ca²⁺-mediated necrotic cell death predominating.     

Characterization of the expression and inheritance of potato leafroll virus (PLRV) and potato virus Y (PVY) resistance in three generations of germplasm derived from Solanum etuberosum

Potato virus Y (PVY) and potato leafroll virus (PLRV) are two of the most important viral pathogens of potato. Infection of potato by these viruses results in losses of yield and quality in commercial production and in the rejection of seed in certification programs. Host plant resistance to these two viruses was identified in the backcross progeny of a Solanum etuberosum Lindl. somatic hybrid. Multiple years of field evaluations with high-virus inoculum and aphid populations have shown the PVY and PLRV resistances of S. etuberosum to be stably expressed in two generations of progeny. However, while PLRV resistance was transmitted and expressed in the third generation of backcrossing to cultivated potato (Solanum tuberosum L. subsp. tuberosum), PVY resistance was lost. PLRV resistance appears to be monogenic based on the inheritance of resistance in a BC₃ population. Data from a previous evaluation of the BC₂ progeny used in this study provides evidence that PLRV resistance was partly conferred by reduced PLRV accumulation in foliage. The field and grafting data presented in this study suggests that resistance to the systemic spread of PLRV from infected foliage to tubers also contributes to the observed resistance from S. etuberosum. The PLRV resistance contributed by S. etuberosum is stably transmitted and expressed through sexual generations and therefore would be useful to potato breeders for the development of PLRV resistant potato cultivars.     

Arachidonic Acid Inhibition of Muscarinic Receptor-Mediated Nitric Oxide Production Occurs at the Level of Calcium Mobilization in Chinese Hamster Ovary Cells

Strong evidence supports that nitric oxide (NO) alters cell signaling pathways involving arachidonic acid (AA). Little is known, however, about the reciprocal modulation of nitrergic pathways by AA. The effects of exogenous AA on signal transduction of M₁ muscarinic acetylcholine receptors were investigated in a model system of stably transfected Chinese hamster ovary cells. AA concentration-dependently inhibited the effects of carbachol in producing NO (IC₅₀ = 191 M) but did not alter inositol phosphate production or M₁ receptor binding. AA inhibited both carbachol-induced transient and sustained increase in intracellular calcium concentration ([Ca²⁺]_i; IC₅₀ = 11 and 12 M, respectively). Furthermore, AA-induced increase in [Ca²⁺]_i cross-desensitizes with thapsigargin, but AA does not inhibit Ca²⁺-ATPase activity. These data support the concept that AA concentration-dependently inhibits receptor-mediated NO production at the level of calcium mobilization.     

Alpha-momorcharin enhances Nicotiana benthamiana resistance to tobacco mosaic virus infection through modulation of reactive oxygen species

Alpha-momorcharin (α-MMC), a member of the plant ribosomal inactivating proteins (RIPs) family, has been proven to exhibit important biological properties in animals, including antiviral, antimicrobial, and antitumour activities. However, the mechanism by which α-MMC increases plant resistance to viral infections remains unclear. To study the effect of α-MMC on plant viral defence and how α-MMC increases plant resistance to viruses, recombinant DNA and transgenic technologies were employed to investigate the role of α-MMC in Nicotiana benthamiana resistance to tobacco mosaic virus (TMV) infection. Treatment with α-MMC produced through DNA recombinant technology or overexpression of α-MMC mediated by transgenic technology alleviated TMV-induced oxidative damage and reduced the accumulation of reactive oxygen species (ROS) during TMV-green fluorescent protein infection of N. benthamiana. There was a significant decrease in TMV replication in the upper leaves following local α-MMC treatment and in α-MMC-overexpressing plants relative to control plants. These results suggest that application or overexpression of α-MMC in N. benthamiana increases resistance to TMV infection. Finally, our results showed that overexpression of α-MMC up-regulated the expression of ROS scavenging-related genes. α-MMC confers resistance to TMV infection by means of modulating ROS homeostasis through controlling the expression of antioxidant enzyme-encoding genes. Overall, our study revealed a new crosstalk mechanism between α-MMC and ROS during resistance to viral infection and provides a framework to understand the molecular mechanisms of α-MMC in plant defence against viral pathogens.     

2-Deoxyglucose resistance: a novel selection marker for plant transformation

A novel selection marker for plant transformation alternative to antibiotic and herbicide resistance is described. The selective agent applied is 2-deoxyglucose (2-DOG) which in the cytosol of plant cells is phosphorylated by hexokinase yielding 2-DOG-6-phosphate (2-DOG-6-P). 2-DOG-6-P exerts toxic effects on overall cellular metabolism leading to cell death. We observed that constitutive expression of the yeast DOG ^R1 gene encoding a 2-DOG-6-P phosphatase resulted in resistance towards 2-DOG in transgenic tobacco plants. This finding was exploited to develop a selection system during transformation of tobacco and potato plants. The lowest concentration of 2-DOG leading to nearly complete inhibition of regeneration of wild-type explants was found to range between 400 and 600 mg/l 2-DOG for tobacco, potato and tomato plants. After Agrobacterium tumefaciens-mediated transformation cells expressing the DOG ^R1 gene were selected by resistance to 2-DOG. More than 50% of tobacco explants formed shoots and on average 50% of these shoots harboured the DOG ^R1 gene. Similar results were obtained for potato cv. Solara. The acceptability of the resistance gene derived from baker's yeast, the unobjectionable toxicological data of 2-DOG as well as the normal phenotype of DOG ^R1-expressing plants support the use of this selection system in crop plant transformation.     

Plant Resistance Suppressors in the Pathosystem Formed by Potato and the Causal Agent of Late Blight

The properties and effects of two plant resistance suppressors (1,3--1,6--glucan and a pentasaccharide of xyloglucan origin) involved in the pathosystem of potato (Solanum tuberosum) and the causal agent of blight (Phytophthora infestans(Mont) de Bary) were compared. The microbial 1,3--1,6--glucan suppressed the defense response over a narrow concentration range (10^–2M), whereas the plant pentasaccharide had a broad range of effective concentrations (10^–12to 10^–6M). In the pathosystem of potato and the causal agent of late blight, the -glucan caused a local and race-specific suppressor effect on the plant host defense response. In contrast, the pentasaccharide caused both local and systemic suppression of potato resistance and the presence of terminal fucosyl residue in the xyloglucan oligosaccharine played a decisive role in its effect. The recognition of both suppressors by potato cell membrane sites is discussed.     

In vitro Plant Formation from Stem Explants of Rape (Brassica napus cv. Zephyr)

Internodal segments from 6-weeks-old rape plants (Brassica napus L. cv. Zephyr) were induced to differentiate in vitro producing shoots or shoots and roots on synthetic nutrient medium under controlled conditions. Benzyladenine (BA) alone (5 × 10^?6 M) induced multiple shoot formation on all stem explants. Roots were induced on shoots when recultured on nutrient medium supplemented with auxins such as naphthalene-acetic acid (NAA) or indoleacetic acid (1AA) or when planted in vermiculite. Complete plant formation was obtained when NAA (2 × 1^?6, 5 × 10^?6 and 10^?5 M) was employed in conjunction with BA at 5 × 10^?6M. At higher concentrations (10^?5M) NAA retards the shoot development while 1AA suppresses it totally. Lower levels of auxins along with the cytokinin did not retard or inhibit shoot differentiation.     

Arachidonic acid inhibits capacitative Ca2+ entry and activates non-capacitative Ca2+ entry in cultured astrocytes

Arachidonic acid (AA) plays important physiological or pathophysiological roles. Here, we show in cultured rat astrocytes that: (i) endothelin-1 or thapsigargin (Tg) induces store-depleted activated Ca²⁺ entry (CCE), which is inhibited by 2-aminoethoxydiphenyl borane (2-APB) or La³⁺; (ii) AA (10 μM) and other unsaturated fatty acids (8,11,14-eicosatrienoic acid and γ-linoleic acid) have an initial inhibitory effect on the CCE, due to AA- or fatty acid-induced internal acid load; (iii) after full activation of CCE, AA induces a further Ca²⁺ influx, which is not inhibited by 2-APB or La³⁺, indicating that AA activates a second Ca²⁺ entry pathway, which coexists with CCE; and (iv) Tg or AA activates two independent and co-existing non-selective cation channels and the Tg-induced currents are initially inhibited by addition of AA or weak acids. A possible pathophysiological effect of the AA-induced [Ca]_i overload is to cause delayed cell death in astrocytes.     

High expression level of antioxidants and pharmaceutical bioactivities of endophytic fungus Chaetomium globosum JN711454

In order to maximize antioxidant activity of pharmaceutical bioactive endophytic fungus Chaetomium globosum JN711454 during fermentation process, designed fermentation experiments of culture media for three levels of eight culture factors were performed using a Taguchi orthogonal array (OA) design with layout L18 (2¹ × 3⁷). The agitation and the potato extract were the most significant affecting factors, and their interaction contributed significantly to fungus activity. The production of antioxidants was more favorable for static condition with 25 g potato extract/100 m. The remaining factors had no strong impact when considered individually. The validation of statistically optimized medium indicated the improvement of antioxidant activity to a level of twofold with approximately overall 40% enhancement in activity. The extract of optimized medium was investigated for various pharmaceutical bioactivities; it revealed a moderate antimicrobial activity, strong anticancer activity against HepG-2, UACC62 cell lines, an antiviral activity against HSV-2 virus, and strong inhibitory activity to butyrylcholinesterase enzyme, one of the neurohydrolase enzymes that play a major role in development of Alzheimer’s disease. As a result of applying statistical fermentation designs, the optimized conditions of endophytic fungus C. globosum JN711454 developed a cost-effective production medium by using inexpensive commercial potato extracts statically, which can lower the energy requirement and could become an efficient, economic, and viable fermentation process for production of pharmaceutical secondary metabolites.     

Arachidonic acid stimulates interleukin-6 release from rat peritoneal macrophages in vitro: Evidence for a prostacyclin-dependent mechanism

Interleukin-6 (IL-6) is a cytokine involved in the differentiation of B-cells to antibody secreting plasma cells, the activation of T-cells, and the stimulation of hepatocyte production of acute phase proteins. Because of the pro-inflammatory effects of this cytokine, we investigated the ability of the fatty acid arachidonic acid (AA) to regulate the release of IL-6 from rat resident peritoneal macrophages (Mø) in vitro. AA (0.5–16 μM) stimulated IL-6 release during a 4 h incubation period in a biphasic manner, with 4 μM AA generating a peak of IL-6 release (3-5-fold). AA (0.5–16 μM) also induced an increasing release of the AA metabolite thromboxane B₂ (TXB₂). The AA-induced release of IL-6 occurred within 1–2 h of incubation, whereas TXB₂ concentrations were elevated within 5 min of AA treatment. The TX synthetase inhibitor CGS 12970 (4.0 μM and 40.0 μM) effectively blocked the generation of TXB₂, but increased prostacyclin (PGI₂) generation and potentiated the release of IL-6. In addition, PGI₂, as well as the PGI₂ agonists iloprost and cicaprost, stimulated IL-6 release from Mø by greater than 5-fold over vehicle-treated basal levels. These data suggest that PGI₂ (but not TXA₂) is involved in AA-induced IL-6 release from peritoneal Mø.