Cent Eur J Biol • 7(1) • 2012 • 132-137 DOI: 10.2478/s11535-011-0104-y Central European Journal of Biology Cholic acid changes defense response to oxidative stress in soybean induced by Aspergillus niger Research Article Djordje Malenčić*, Slavko Kevrešan, Milan Popović, Dubravka Štajner, Boris Popović, Biljana Kiprovski, Simonida Djurić Faculty of Agriculture, University of Novi Sad, 21000 Novi Sad, Serbia Received 27 April 2011; Accepted 22 November 2011 Abstract: The oxidative stress and antioxidant systems in soybean leaves and roots infected with plant pathogen Aspergillus niger were studied following treatment with different concentrations of cholic acid Several oxidative stress parameters were analyzed: production of superoxide (O2•-) and hydroxyl radicals (•OH), lipid peroxidation (LP), and superoxide dismutase (SOD; EC 1.15.1.1) activity, as well as the content of reduced glutathione (GSH) Results showed that inoculation with A niger led to the increase of O2•- production and GSH quantities in leaves and •OH in roots The highest activity of SOD occured in infected plants treated with cholic acid in concentrations of 40 and 60 mg L-1 which ultimately led to a decrease in O2•- production Inoculation with Aspergillus in combination with elevated cholic acid concentrations also increased •OH production which is correlated with increased LP These results may support the idea of using cholic acid as an elicitor to trigger hypersensitive response in plant cells Use of cholic acid may also actively contribute to soybean plants defense response against pathogen attack Keywords: Aspergillus niger • Cholic acids • Lipid peroxidation • Oxidative stress • Reactive oxygen species © Versita Sp z o.o Abbreviations: GSH HR LP MDA ROS SOD - reduced glutathione; - hypersensitive response; - lipid peroxidation; - malonyldialdehyde; - reactive oxygen species; - superoxide dismutase Introduction Some species of the genus Aspergillus are recognized as the most widely distributed fungi encountered in foodstuffs, soils and other materials Several Aspergilli produce toxic metabolites (mycotoxins) or cause serious diseases (mycoses) Among these, A flavus produces aflatoxin B1, the most potent naturally occurring carcinogenic substance known [1] Other Aspergilli (A niger, A sojae, A oryzae) are grown in controlled fermentations to produce an assortment of small molecules of industrial importance 132 In plants Aspergilli are known to cause discoloration, seed deterioration and seedling and epicotyl rot Plants have evolved efficient mechanisms to combat pathogen attack One of the first responses to attempted pathogen attack is the generation of a burst of antioxidants that triggers hypersensitive cell death This is called the hypersensitive response (HR) and is considered to be a major element of plant disease resistance The HR is thought to deprive pathogens of food and to confine them to initial infection sites [2] The HR occurs when a plant specifically recognizes a pathogen, during an `incompatible` interaction [3] Cell death manifests as membrane disruption, nucleus condensation, fragmentation of DNA, shrinkage of the cell, leading to tissue collapse Cell death is also a feature of disease symptoms, but in the case of such `compatible` interactions, it occurs very late in the infection process The HR is not due to the action of pathogen virulence factors that kill the plant cells, but rather appears to be a form of programmed cell death (PCD) in plants [4] PCD is initiated by the production of highly reactive oxygen species (ROS) by the oxidative metabolism * E-mail: malencic@polj.uns.ac.rs D Malenčić et al of normal aerobic cells ROS, including superoxide radical (O2.-), hydrogen peroxide (H2O2), hydroxyl radical (.OH), and singlet oxygen (1O2), have been implicated in a number of physiological disorders in plants [5] Free radical formation can be induced by a number of stressors, including UV light and other forms of radiation, photooxidation, air pollution, drought, herbicides, pathogen invasion, certain injuries, hyperoxia, ozone, and temperature fluctuations [6,7] The ubiquity of such stressors require that plants also have a way of protecting themselves from the damaging effects of ROS Antioxidant systems are produced during interactions between pathogens and plant hosts [8,9] Plants possess a number of enzymatic and non-enzymatic mechanisms of detoxification to efficiently scavenge for either the ROS themselves or their secondary reaction products Various enzyme systems participate in ROS metabolism during the pathogen attack in plants Major ROS-scavenging enzymes such as superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), and ascorbate peroxidase (APX) are produced to avoid cellular disintegration by ROS SOD, the first enzyme in ROS metabolism catalyzes dismutation of O2.- and HO2 to H2O2 [10] Cholic acid, one of the bile acids, is an elicitor of hypersensitive cell death, pathogenesis-related protein synthesis, and phytoalexin accumulation in rice plants [11,12] Elicitor molecules trigger a plant defense response which involves the production of ROS [13,14] Bile acids can also promote the generation of ROS The increase in ROS by bile acids has been well documented in mammalian tissues [15,16], but Kevrešan et al [17] has shown that treatment of young soybean plants with cholic acid affects their oxidative status Treatments with cholic acid increased O2.-, LP, OH and GSH in leaves of treated plants while in roots, higher cholic acid concentrations increased OH production but not other ROS Thus, the main objective of this study was to evaluate the effect of cholic acid on several antioxidants in soybean plants exposed to a mild biotic stress induced by infection with A niger Experimental Procedures 2.1 Fungal culture A niger cultures were grown at 25°C in the dark on potato dextrose agar, according to Boue et al [1] Conidia were harvested from 5-day-old cultures of A niger Conidia were suspended in cm3 sterile, distilled water, containing 3.9x109 conidia cm-3 2.2 Plant material Soybean seeds (genotype Bečejka), of unknown susceptibility to this particular A niger isolate, were obtained from the Institute of Field and Vegetable Crops, Novi Sad, Serbia Before treatments seeds were surface sterilized in 5% solution of commercial bleach for 20 min, rinsed with distilled water and sterilized again in 70% ethanol for one minute followed by ten minutes in commercial bleach Seeds were then rinsed three times with sterile distilled water and placed in an incubator for days at 25°C Seedlings were then transferred to experimental chamber in pots with full nutrient solution (1 mM L-1 MgSO4; mM L-1 Ca(NO3)2; 0.19 mM L-1 KH2PO4; 0.31 mM L-1 NH4H2PO4; 46 μM L-1 B; μM L-1 Mn; 0.8 μM L-1 Zn; 0.3 μM L-1 Cu; 0.8 μM L-1 Mo; and 75 μM L-1 Fe as Fe-EDTA) and grown hydroponically in controlled environment (temperature 25°C, relative humidity 60%, light intensity 16000 lux) for two weeks Treatments with different concentrations of cholic acid were carried out by adding cholic acid (as sodium cholate) in nutrient solution at concentrations 20, 40, 60, and 80 mg L-1 A niger treatments were obtained by adding cm3 of inoculum to 700 cm3 of nutrient solution, resulting in a final concentration of 107 conidia cm-3 nutrient solution Control plants were either grown in nutrient solution without addition of cholic acid or A niger (0, first control), or they were grown in nutrient solution without addition of cholic acid, but inoculated with A niger (0+A, second control) Samples of roots and leaves from treatment and control plants were taken for biochemical analyses days after treatments, when plants had four fully expanded leaves (20 days old) 2.3 Biochemical assays For the determination of the oxidative stress parameters, g of fresh plant material was homogenized with 10 cm3 0.1 M K2HPO4 at pH 7.0 After centrifugation at 15000xg for 10 at 4°C, aliquots of the supernatant were used for biochemical assays Levels of superoxide radical were determined by the inhibition of adrenaline autooxidation [18] Hydroxyl radical levels were measured by the inhibition of deoxyribose degradation [19] Superoxide-dismutase (SOD; EC 1.15.1.1) activity was assayed by measuring its ability to inhibit photochemical reduction of nitro blue tetrazolium (NBT) chloride The reaction mixture (4 cm3) contained 63 µM NBT, 13 mM L-methionine, 0.1 mM EDTA, 13 µM riboflavin, 0.05 M sodium carbonate and 0.5 cm3 enzyme extract (0.5 cm3 distilled water in case of control) The mixture was incubated under two 15 W fluorescent lamps for 15 at 25°C, followed by transfer to dark for 15 min, after which the absorbance 133 Cholic acid changes defense response to oxidative stress in soybean induced by Aspergillus niger was read at 560 nm One unit of the SOD activity was defined as the amount of enzyme required to inhibit reduction of NBT by 50% [10] Lipid peroxidation (LP) was measured in terms of malonyldialdehyde (MDA) content, a thiobarbituric acid reactive substance as described by Placer et al [20] and Gidrol et al [21] For this assay, plant material was first homogenized and then extracted in 0.1% trichloroacetic acid (TCA) in ratio 1:5 (w/v) and centrifuged at 12000xg for 30 at 4°C One cm3 of supernatant was incubated with cm3 20% TCA containing 0.5% thiobarbituric acid for 30 at 95°C The reaction was stopped by cooling on ice for 10 and the product was centrifuged at 10000xg for 15 The absorbance of the reaction product was measured at 532 nm MDA concentration was determined using the extinction coefficient of 155 mM-1 cm-1 and expressed as µM g-1 FW Reduced glutathione (GSH) was determined using Ellman reagent [22] and expressed as µmol GSH g-1 FW were no significant changes in GSH in either roots or leaves treated with cholic acid and A niger (Figure 2B) Discussion One of the earliest biochemical changes observed after pathogen recognition is an increased production of ROS, in a so-called `oxidative burst` Numerous reports show a rapid production of ROS in response to a range of infections or elicitor treatments [23-25] Superoxide radical and H2O2 influence the signal transduction pathway leading to HR cell death by acting directly as antimicrobial compounds or by inducing the rapid cross- 2.4 Statistical analyses The experiment was replicated three times With each replication, assays were performed in triplicate Values are the mean ± standard deviation of the three replicates Statistical significance was tested with a one-way ANOVA followed by comparisons of means by Duncan’s multiple range test (P