Detection of Lipid Peroxidation-Derived Free Azelaic Acid, a Biotic Stress Marker and Other Dicarboxylic Acids in Tobacco by Reversed-Phase HPLC-MS Under Non-derivatized Conditions.

Azelaic acid (AzA, 1,9-nonadienoic acid) is a nine-carbon chain (C9) dicarboxylic acid with multiple and diverse functions in humans and plants. In plants this compound was suggested as a marker for lipid peroxidation under biotic and abiotic stress conditions and an inducer (priming agent) of plant immunity (acquired resistance). Detection methods for AzA in plants include a wide range of methodological approaches. This new and simple reversed-phase HPLC-MS protocol describes the measurement of AzA and other dicarboxylic acids either from tobacco leaf tissue or petiolar exudates (vascular sap) of plants under non-derivatized conditions.

Glutathione Can Compensate for Salicylic Acid Deficiency in Tobacco to Maintain Resistance to Tobacco Mosaic Virus.

Earlier studies showed that the artificial elevation of endogenous glutathione (GSH) contents can markedly increase the resistance of plants against different viruses. On the other hand, salicylic acid (SA)-deficient NahG plants display enhanced susceptibility to viral infections. In the present study, the biochemical mechanisms underlying GSH-induced resistance were investigated in various tobacco biotypes displaying markedly different GSH and SA levels. The endogenous GSH levels of Nicotiana tabacum cv. Xanthi NN and N. tabacum cv. Xanthi NN NahG tobacco leaves were increased by infiltration of exogenous GSH or its synthetic precursor R-2-oxo-4-thiazolidine-carboxylic acid (OTC). Alternatively, we also used tobacco lines containing high GSH levels due to transgenes encoding critical enzymes for cysteine and GSH biosynthesis. We crossed Xanthi NN and NahG tobaccos with the GSH overproducer transgenic tobacco lines in order to obtain F1 progenies with increased levels of GSH and decreased levels of SA. We demonstrated that in SA-deficient NahG tobacco the elevation of in planta GSH and GSSG levels either by exogenous GSH or by crossing with glutathione overproducing plants confers enhanced resistance to Tobacco mosaic virus (TMV) manifested as both reduced symptoms (i.e. suppression of hypersensitive-type localized necrosis) and lower virus titers. The beneficial effects of elevated GSH on TMV resistance was markedly stronger in NahG than in Xanthi NN leaves. Infiltration of exogenous GSH and OTC or crossing with GSH overproducer tobacco lines resulted in a substantial rise of bound SA and to a lesser extent of free SA levels in tobacco, especially following TMV infection. Significant increases in expression of pathogenesis related (NtPR-1a, and NtPRB-1b), and glutathione S-transferase (NtGSTtau, and NtGSTphi) genes were evident in TMV-inoculated leaves in later stages of pathogenesis. However, the highest levels of defense gene expression were associated with SA-deficiency, rather than enhanced TMV resistance. In summary, elevated levels of glutathione in TMV-infected tobacco can compensate for SA deficiency to maintain virus resistance. Our results suggest that glutathione-induced redox changes are important components of antiviral signaling in tobacco.

Comparing the agrochemical properties of compost and vermicomposts produced from municipal sewage sludge digestate.

The aim of this work was to investigate whether the agronomic traits of vermicompost prepared from partially stabilised sewage sludge digestate after thermophilic composting were more favourable than those of conventional compost. The effects of various additives (green waste, spent mushroom compost, wheat straw, biochar) were also tested after 1.5 months precomposting followed by 3 months vermicomposting with Eisenia fetida or by compost maturing. Vermicomposting did not result in significantly more intensive mineralisation than composting; the average organic carbon contents were 21.2 and 22.2% in vermicomposts and composts, respectively. Hence, the average total (N: 2.4%; P: 1.9%; K: 0.9%) and available (N: 160 mg/kg; P: 161 mg/kg; K: 0.8%) macronutrient concentrations were the same in both treatments. The processing method did not influence the organic matter quality (E4/E6) either. However, on average the concentration of the plant growth regulator kinetin was more than twice as high in vermicomposts.

Signals of Systemic Immunity in Plants: Progress and Open Questions.

Systemic acquired resistance (SAR) is a defence mechanism that induces protection against a wide range of pathogens in distant, pathogen-free parts of plants after a primary inoculation. Multiple mobile compounds were identified as putative SAR signals or important factors for influencing movement of SAR signalling elements in Arabidopsis and tobacco. These include compounds with very different chemical structures like lipid transfer protein DIR1 (DEFECTIVE IN INDUCED RESISTANCE1), methyl salicylate (MeSA), dehydroabietinal (DA), azelaic acid (AzA), glycerol-3-phosphate dependent factor (G3P) and the lysine catabolite pipecolic acid (Pip). Genetic studies with different SAR-deficient mutants and silenced lines support the idea that some of these compounds (MeSA, DIR1 and G3P) are activated only when SAR is induced in darkness. In addition, although AzA doubled in phloem exudate of tobacco mosaic virus (TMV) infected tobacco leaves, external AzA treatment could not induce resistance neither to viral nor bacterial pathogens, independent of light conditions. Besides light intensity and timing of light exposition after primary inoculation, spectral distribution of light could also influence the SAR induction capacity. Recent data indicated that TMV and CMV (cucumber mosaic virus) infection in tobacco, like bacteria in Arabidopsis, caused massive accumulation of Pip. Treatment of tobacco leaves with Pip in the light, caused a drastic and significant local and systemic decrease in lesion size of TMV infection. Moreover, two very recent papers, added in proof, demonstrated the role of FMO1 (FLAVIN-DEPENDENT-MONOOXYGENASE1) in conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates after microbial attack and acts as a potent inducer of plant immunity to bacterial and oomycete pathogens in Arabidopsis. These results argue for the pivotal role of Pip and NHP as an important signal compound of SAR response in different plants against different pathogens.

BioArena system for knowing and understanding the biological world: a review with new experimental results.

A simple observation is the basis of the development of BioArena system: according to the first observations during the biological incubation after inoculation there is formaldehyde (HCHO) emission from the chromatographic spots; in this emission process, the level of HCHO molecules decreases time dependently. In fact, the antibiotic effect of an antibiotic-like compound decreases in parallel with the HCHO emission. The investigations demonstrated clearly a unique function and role of endogenous HCHO and its one main reaction product, ozone (O3), in the antiproliferative (e.g., antimicrobial) effect of different molecules with diverse chemical structures. The results in BioArena can be extended for in vivo conditions (e.g., greenhouse experiments), as well. For the pretreatment with different doses of inducers (immunostimulation-inducing molecules) there are always four bioequivalent immunostimulating response ranges (quadruple bioequivalent immune response system) in plants. The inducers (e.g., N-methylated basic amino acids, salicylic acid, cinnamic acid, and trace elements) do not participate directly in the induction of the immunostimulating effect. These new findings support a statement that HCHO and its reaction products (mainly O3), as bioreactive small molecules, are responsible for the immunostimulating activity (in vivo conditions), as well.

Effect of ascorbigen and 1'-methylascorbigen on disease resistance of bean plants to Uromyces phaseoli.

The effect of ascorbigen and 1'-methylascorbigen on the disease resistance of bean (Phaseolus vulgaris L.,. cv. Debreceni Tarka) to the fungal pathogen Uromyces phaseoli Pers. is reported. Contrary to ascorbigen, pretreatment of bean plants with l'-methylascorbigen, as in the case of other endogenous N-, O- and S-methyl compounds, induced the double immune response leading to the biochemical immunization of plants. The effectiveness of protection depended on the dosage of the applied l'-methylascorbigen and on the time interval between the chemical pretreatment and inoculation. Results of our greenhouse experiments revealed, in accordance with previous results, that the presence of the N-methyl group in the 1'-methylascorbigen molecule is the precondition of the effect, and formaldehyde formed from this N-methyl group and its reaction products (e.g. singlet oxygen, ozone) can have a determining role in the manifestation of the effect.

Potential role of formaldehyde in the mechanism of action of ascorbigens on the basis of BioArena studies.

The effect of ascorbigen and 1'-methylascorbigen as a model compound pair was studied on the phytopathogenic bacterium Pseudomonas savastanoi pv. phaseolicola in the BioArena experimental system after overpressured layer chromatography. Results showed a characteristic, strong antibacterial effect of 1'-methylascorbigen and weak effect of ascorbigen present on the adsorbent layer as chromatographic spot. Addition of formaldehyde capture compounds (L-arginine, glutathione, dimedone) partially or totally reduced the antibacterial effect of 1'-methylascorbigen and ascorbigen. On adding Cu(II) ions--which mobilize and coordinate formaldehyde--to the culture medium, the antibacterial effect of both compounds became stronger. It is supposed that the weak antibacterial effect of ascorbigen may have originated from the 1'-methylascorbigen formed in situ on the adsorbent layer by partial enzymatic methylation of ascorbigen.

[Role of formaldehyde and its reaction products in mechanism of action of some plant ingredients]. / A formaldehid és reakciótermékeinek szerepe a növényi hatóanyagok hatásmechanizmusában.

BioArena as a complex bioautographic system was successfully used for studying mechanism of action of some characteristic plant ingredients (e.g., 1'-methyl-ascorbigen, trans-resveratrol, salicylic acid, cinnamic acid). It has been established that the formaldehyde as a main component of formaldehyde cycle and other biochemical pathways can be studied in chromatographic spots as well. Ozone as a specially reactive reaction product of formaldehyde can be measured indirectly there. Different formaldehyde-capturing and -giving molecules and other compounds influenced (inhibited or promoted) characteristically the antibiotic effect.

Thin-layer chromatography-densitometry and liquid chromatography analysis of alkaloids in leaves of Papaver somniferum under stress conditions.

The effect of stress conditions on the concentrations of secondary metabolites were examined during various developmental stages of Papaver somniferum plants. P. somniferum plants were grown in laboratory conditions (Budakalász). The experiment consisted of 22 treatments. Significantly different alkaloid contents can be observed under different stress conditions. In general, the alkaloid contents of plants are very low; therefore, a highly sensitive and reliable method has to be developed for analysis. The amount of alkaloids was measured by 2 separation and detection techniques. Accuracy of the thin-layer chromatography method for quantitative analysis is limited. Without purification of samples the background is too noisy. Column liquid chromatography is a sensitive and relatively inexpensive method that allows precise quantitative determination of the alkaloid content.

Possible Role of Formaldehyde in the Apoptotic and Mitotic Effect of 1-Methyl-Ascorbigen.

PC-3 human prostate carcinoma cells were treated with 100 mg/ml 1-methyl-ascorbigen (Me-Asc). This treatment resulted in a significant decrease in tumor cell number in parallel with an increase in apoptotic cells. The formaldehyde (HCHO) level in the culture medium was also increased. Dimedone (Di), a known capture molecule forming formal-demethone with HCHO, applied simultaneously with Me-Asc in 10 mg/ml doses diminished the apoptosis-inducing effect of Me-Asc. The possible role of in situ generated HCHO in the induction of apoptosis is discussed.