Gábor Gullner

Salt tolerance of barley induced by the root endophyte Piriformospora indica is associated with a strong increase in antioxidants.

The root endophytic basidiomycete Piriformospora indica has been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. Biochemical mechanisms underlying P. indica-mediated salt tolerance were studied in barley (Hordeum vulgare) with special focus on antioxidants. Physiological markers for salt stress, such as metabolic activity, fatty acid composition, lipid peroxidation, ascorbate concentration and activities of catalase, ascorbate peroxidase, dehydroascorbate reductase, monodehydroascorbate reductase and glutathione reductase enzymes were assessed. Root colonization by P. indica increased plant growth and attenuated the NaCl-induced lipid peroxidation, metabolic heat efflux and fatty acid desaturation in leaves of the salt-sensitive barley cultivar Ingrid. The endophyte significantly elevated the amount of ascorbic acid and increased the activities of antioxidant enzymes in barley roots under salt stress conditions. Likewise, a sustained up-regulation of the antioxidative system was demonstrated in NaCl-treated roots of the salt-tolerant barley cultivar California Mariout, irrespective of plant colonization by P. indica. These findings suggest that antioxidants might play a role in both inherited and endophyte-mediated plant tolerance to salinity.

Local and Systemic Responses of Antioxidants to Tobacco Mosaic Virus Infection and to Salicylic Acid in Tobacco (Role in Systemic Acquired Resistance).

Changes in ascorbate and glutathione levels and in activities of ascorbate peroxidase, catalase, dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST), and superoxide dismutase (SOD) were investigated in tobacco mosaic virus (TMV)-inoculated lower leaves and in non-inoculated upper leaves of Nicotiana tabacum L. cv Xanthi-nc. In separate experiments the effects of exogenous salicylic acid (SA) were also studied. Symptom appearance after TMV inoculation was preceded by a slight, transient decline of ascorbate peroxidase, GR, GST, and SOD activities in the inoculated lower leaves, but after the onset of necrosis these activities and the glutathione level substantially increased. Ascorbic acid level and DHAR activity declined and dehydroascorbate accumulated in the inoculated leaves. In upper leaves, the glutathione level and the activities of GR, GST, and SOD increased 10 to 14 d after TMV inoculation of the lower leaves, concomitantly with the development of systemic acquired resistance. From the six distinct SOD isoenzymes found in tobacco leaves, only the activities of Cu,Zn-SOD isoenzymes were affected by TMV. SA injection induced DHAR, GR, GST, and SOD activities. Catalase activities were not modified by TMV infection or SA treatment. It is supposed that stimulated antioxidative processes contribute to the suppression of necrotic symptom development in leaves with systemic acquired resistance.

Endophytic Burkholderia fungorum DBT1 can improve phytoremediation efficiency of polycyclic aromatic hydrocarbons

Burkholderia fungorum DBT1 is a bacterial strain isolated from an oil refinery discharge and capable of transforming dibenzothiophene, phenanthrene, naphthalene, and fluorene. In order to evaluate the influence of a policyclic aromatic hydrocarbon (PAH)-transforming bacterial strain on the phytoremediation of organic contaminants, B. fungorum DBT1 was inoculated into hybrid poplar (Populus deltoides×Populus nigra). The poplar plants were grown for 18-wk with or without naphthalene, phenanthrene, fluorene and dibenzothiophene (488mgkg(-1) soil each) in non-sterile sand-peat substrate. Evidences were gained that B. fungorum DBT1 was present in high concentration in poplar root tissues (2.9-9.5×10(3)CFUg(-1)), while the strain was not detected in stem, leaves and rhizosphere. When poplar was planted in uncontaminated substrate, the infection caused negative effects on biomass index, leaves and stem dry weight, without showing however any disease symptoms. On the other hand, plants inoculated with the strain DBT1 resulted in better tolerance against the toxic effects of PAHs, in terms of root dry weight. Although the presence of plants acted as the main effective treatment for PAH dissipation (82-87%), the inoculum with DBT1 strain lead to the highest PAH abatement (up to 99%). In the present study, an environmental isolate with proper metabolic features was demonstrated to be possibly suitable as a poplar endophyte for improving microbe-assisted phytoremediation in PAH contaminated matrices.

Elevation of glutathione level and activation of glutathione-related enzymes affect virus infection in tobacco.

The effects of two chemicals, L-2-oxothiazolidine-4-carboxylic acid (OTC) and (S)-carvone, were investigated on the development of necrotic symptoms and on the virus concentration in tobacco mosaic virus (TMV)-infected tobacco plants. OTC treatments markedly increased the cellular glutathione (GSH) levels in tobacco leaf discs. In addition, OTC pretreatment considerably decreased both the number of necrotic lesions and the virus content in TMV-infected leaf discs. The monoterpene (S)-carvone increased only slightly the GSH content of leaf tissues and caused lipid peroxidation. (S)-carvone dramatically induced the activity of glutathione S-transferase and to a lesser extent elevated also the activities of ascorbate peroxidase and glutathione reductase. Treatments with (S)-carvone strongly reduced the number and size of necrotic lesions, but did not influence the virus concentration. The results show that increased levels of GSH and activities of GSH-related enzymes by OTC and (S)-carvone reduce necrotization of virus-infected tissues. However, virus multiplication and lesion formation do not necessarily correlate: virus multiplication is suppressed only by substantially elevated GSH contents.

Effects of brassinosteroid infiltration prior to cold treatment on ion leakage and pigment contents in rape leaves

The effect of 24-epibrassinolide (BR27) on cold resistance of rape seedlings was studied by ion leakage and photosynthetic pigment degradation measurements. Aqueous solutions of BR27 were injected into cotyledons or primary leaves of rape plants and these plants were incubated at 2 °C or 20 °C. Cold treatment (2 °C) without BR27 injection elevated the membrane permeability in both primary leaves and cotyledons significantly. Surprisingly, injection of leaves with water or 0.467 % aqueous ethanol solution led to a massive increase in membrane permeability after cold stress at 2 °C. The synergistic effect of leaf infiltration and cold on permeability was abolished by 0.05 and 1.00 µM of BR27 in primary leaves and by 1.00 µM of BR27 in cotyledons. On the other hand, BR27 solutions strongly elevated the membrane permeability at 20 °C, while water and ethanol solutions brought about only negligible increases. Water or ethanol infiltrations strongly reduced the leaf contents of chlorophyll (Chl) a, Chl b and carotenoids at 2 °C but less markedly at 20 °C. However, in seedlings exposed to 2 °C pigments content was significantly higher in BR27-treated leaves as compared to water/ethanol control. There were no differences between pigment contents of leaves injected with BR27 solutions or only water/ethanol at 20 °C. The above data strongly support the stress protecting effect of BR27.

Sulfate supply influences compartment specific glutathione metabolism and confers enhanced resistance to Tobacco mosaic virus during a hypersensitive response

Sufficient sulfate supply has been linked to the development of sulfur induced resistance or sulfur enhanced defense (SIR/SED) in plants. In this study we investigated the effects of sulfate (S) supply on the response of genetically resistant tobacco (Nicotiana tabacum cv. Samsun NN) to Tobacco mosaic virus (TMV). Plants grown with sufficient sulfate (+S plants) developed significantly less necrotic lesions during a hypersensitive response (HR) when compared to plants grown without sulfate (−S plants). In +S plants reduced TMV accumulation was evident on the level of viral RNA. Enhanced virus resistance correlated with elevated levels of cysteine and glutathione and early induction of a Tau class glutathione S-transferase and a salicylic acid-binding catalase gene. These data indicate that the elevated antioxidant capacity of +S plants was able to reduce the effects of HR, leading to enhanced virus resistance. Expression of pathogenesis-related genes was also markedly up-regulated in +S plants after TMV-inoculation. On the subcellular level, comparison of TMV-inoculated +S and −S plants revealed that +S plants contained 55–132 % higher glutathione levels in mitochondria, chloroplasts, nuclei, peroxisomes and the cytosol than −S plants. Interestingly, mitochondria were the only organelles where TMV-inoculation resulted in a decrease of glutathione levels when compared to mock-inoculated plants. This was particularly obvious in −S plants, where the development of necrotic lesions was more pronounced. In summary, the overall higher antioxidative capacity and elevated activation of defense genes in +S plants indicate that sufficient sulfate supply enhances a preexisting plant defense reaction resulting in reduced symptom development and virus accumulation.

AFLP analysis and improved phytoextraction capacity of transgenic gshI-poplar clones (Populus x canescens L.) for copper in vitro

Abstract Clone stability and in vitro phytoextraction capacity of vegetative clones of P. x canescens (2n = 4x = 38) including two transgenic clones (ggs11 and lgl6) were studied as in vitro leaf disc cultures. Presence of the gshI-transgene in the transformed clones was detected in PCR reactions using gshI-specific primers. Clone stability was determined by fAFLP (fluorescent amplified DNA fragment length polymorphism) analysis. In total, 682 AFLP fragments were identified generated by twelve selective primer pairs after EcoRIDMseI digestion. Four fragments generated by EcoAGTDMseCCC were different (99.4% genetic similarity) which proves an unexpectedly low bud mutation frequency in P. \ canescens. For the study of phytoextraction capacity leaf discs (8 mm) were exposed to a concentration series of ZnSO4 (10-1 to 10-5 ᴍ) incubated for 21 days on aseptic tissue culture media WPM containing 1 μᴍ Cu. Zn2+ caused phytotoxicity only at high concentrations (10-1 to 10-2 ᴍ). The transgenic poplar cyt-ECS (ggs11) clone, as stimulated by the presence of Zn, showed elevated heavy metal (Cu) uptake as compared to the non-transformed clone. These results suggest that gshI-transgenic poplars may be suitable for phytoremediation of soils contaminated with zinc and copper.

Enhanced Inducibility of Antioxidant Systems in a Nicotiana tabacum L. Biotype Results in Acifluorfen Resistance

Abstract Levels of non-protein thiols (mostly glutathione, GSH), ascorbic acid (AA), and activities of the enzymes ascorbate peroxidase (AP), glutathione reductase (GR) and GSH S-transferase (GST) were determined in cell-free leaf extracts of acifluorfen-resistant and -sensitive tobacco plants. These parameters were examined also in detached leaves of the above plants exposed to acifluorfen stress. In leaves of untreated plants the AA content was by 40% higher in the resist ant biotype as compared to the sensitive ones, but the levels of GSH, AP, GR, and GST did not differ significantly in the two biotypes. However, in the resistant leaves stressed by acifluorfen the activity of AP readily increased while in the sensitive leaves it did not change. The levels of GSH and the activities of GR and GST markedly increased in both biotypes after acifluorfen stress, but the induction in the resistant leaves was consistently stronger in each case. The AA contents were increased equally in both biotypes. These parameters were much less affected by paraquat stress. The only significant changes were observed at low concentrations of this herbicide (8 x 10-9 м): when the thiol content and the activity of GST increased in the resistant leaves. Enhanced inducibility of antioxidant systems seems to be involved in resistance of tobacco to acifluorfen stress.

Ability of Poplar (Populus spp.) to Detoxify Chloroacetanilide Herbicides

The ability of poplar trees to resist chemical stress caused by chloroacetanilide herbicides was studied. Detached leaves of Lombardy poplar (Populus nigra L.) were exposed to seven chloroacetanilide herbicides via uptake through the cut petiole. The leaves showed high tolerance against the phytotoxicity of these compounds. Their tolerance was further enhanced by treatment with 2-oxothiazolidine-4-carboxylic acid (OTC, a precursor of the amino acid cysteine). High levels of glutathione (GSH) and GSH S-transferase (GST) activity were detected in poplar leaves. Treatments with chloroacetanilide herbicides left the GSH content in the leaves unchanged but strongly induced the GST activity. In contrast, in OTC-treated leaves increased GSH contents were measured, but GST activities remained unchanged. It therefore appears that a highly active and inducible GSH-conjugation Phase II detoxification system in their leaves may make poplar trees useful for phytoremediation of soils contaminated with chloroacetanilide herbicides. Based on these findings a phytoremediation project using different poplar hybrids at a site heavily polluted with such compounds in Hungary has been launched.

Temperature-dependent formaldehyde metabolism in bean plants. The heat shock response

Abstract A strong correlation exists between the external temperature and the measurable amount of formaldehyde in Pinto bean leaf tissues. The highest amount was detected after the heat shock (HS) treatment of leaves. On the other hand, as a result of HS, the level of some potential formaldehyde generators (trigonelline, choline and N e - trimethyl- l -lysine ) moderately decreased in comparison to the normal room temperature (20°C). In the presence of dimedone the amount of these formaldehyde generators further decreased at all temperatures applied. In one case it was shown that about 4.5% of the radioactivity of Ne-[methyl-3H] trimethyl- l -lysine indeed accumulated in formaldemethone, indicating that N-methylated compounds could be in vivo precursors (generators) of formaldehyde in bean leaf tissues. It is proposed that formaldehyde may play a role in a dynamic demethylation-methylation process that may include also the methylation of heat shock proteins (HSPs).