Concentration-dependent effects of effusol and juncusol from Juncus compressus on Arabidopsis thaliana seedling development
Plant material, growing conditions and treatments
The Arabidopsis thaliana The Columbia-0 ecotype was used for the experiments. The seeds were provided by Edit Horváth (Department of Plant Biology, University of Szeged). The seeds were surface sterilized with a solution of 70% (v/v) ethanol and 20% (v/v) bleach. After washing with distilled water, the seeds were stored at 4°C for 1 day. The seedlings were grown in the greenhouse of the Department of Plant Biology of the University of Szeged. Seeds were planted on 0.5 × Murashige and Skoog agar medium (0.8%)34 with the addition of 0.5% (w/v) sucrose (pH adjusted to 5.5 with NaOH) in round plastic petri dishes (90 x 17 mm) with five seeds per petri dish on a single line. The experimental setup was designed as described by Marik et al.18, and the Petri dishes were positioned vertically. Plants were grown for 5 days in a controlled environment under 200 µmol m−2 s−1 photon flux density (F36W/GRO lamps, OSRAM SYLVANIA, Danvers, MA, USA) with a light/dark period of 12/12 h, day/night temperatures of 24/22°C and relative humidity of 55 to 60%31. Five millimeter holes were drilled in agar with a sterile cork borer 0.5 cm from the end of the 5-day-old roots Arabidopsis sowing. The treatments were carried out by filling the hole with 10 μL of three different concentrations (0, 1, 0, 5 and 1 mM) of Phe, effusol and juncusol dissolved in methanol (Fig. 1). Effusol and juncusol were provided by Andrea Vasas (Faculty of Pharmacy, University of Szeged) and Phe was purchased from Merck Millipore (Darmstadt, Germany).
For control plant treatments, methanol was pipetted into the holes. Sampling of the whole seedling was carried out after 1 week of treatment.
Growth parameters of Arabidopsis thaliana
The biomass of the plants grown with the treatment compounds was measured by an analytical balance (Adam Equipment NBL2541, Milton Keynes, UK) and the samples were stored at -20°C for biochemical analyses. The growth of A. thaliana seeding was monitored by image capture (Canon EOS 700D, Canon, UK). Petri dishes were photographed without a lid on top using a homogeneous surface. Petri dish images were saved in jpeg format, then leaf areas and root lengths were analyzed by ImageJ software ver. 1.52a (National Institute of Mental Health, Bethesda, Maryland, USA) (http://imagej.nih.gov/ij)35. The experiments were repeated three times.
Determination of photosynthetic pigment content from Arabidopsis sowing
Whole plant fresh weights from each plate were measured and photosynthetic pigments were quantified as described by Faragó et al.36. Fresh tissue was ground with ethanol and the homogenate was centrifuged (Eppendorf 5424R Centrifuge, Eppendorf GMBH, Germany) at 12,000 rpm and 4°C for 10 min. The optical densities of the supernatants were detected by a plate reader (Synergy HTX plate reader, BioTek Instruments, Winooski, VT, USA) at 664, 648 and 470 nm. Calculations for chlorophyll-a, chlorophyll-b and carotenoids were described by Faragó et al.36and pigment levels were normalized to 1 g fresh weight.
Determination of total soluble protein content
Soluble proteins were extracted from 100 mg of frozen seedling samples. Homogenization was carried out by Bradford’s method.37. Samples were ground in ice-cold phosphate buffer (KH2Purchase order4 and Na2HPO4, 50 mM, pH 7.0) and centrifuged in an Eppendorf centrifuge (Eppendorf 5424R, Eppendorf GMBH, Germany) for 10 min at 4°C. The supernatant was used to measure total soluble protein content by a 595 nm plate reader (Synergy HTX plate reader, BioTek Instruments, Winooski, VT, USA).
Hydrogen peroxide (H2O2) content measures
The H2O2 levels of Arabidopsis seedlings were measured as described by Horváth et al.34. One hundred milligrams of sample was homogenized in 0.5 ml ice-cold 0.1% TCA. After centrifugation, 0.25 ml of the supernatant was diluted with 0.25 ml of 50 mM potassium phosphate buffer (pH 7.0) and 0.5 ml of 1 M potassium iodide (KI) (in phosphate buffer 50 mM potassium, pH 7.0). The reaction was started with the addition of KI, and the sample was incubated for 10 min at 25°C. Absorbance values were recorded by a spectrophotometer (KONTRON, Milan, Italy) at 390 nm. A standard curve was prepared using the H2O2 Standard. The results were expressed in μmol H2O2 g−1 FW.
Determination of antioxidant enzymatic activities
Enzyme extracts were prepared as described by Horváth et al.34. Measurement of SOD activity (EC 184.108.40.206) was based on the ability of the enzyme to inhibit the photochemical reduction of p-nitro-blue tetrazolium (Sigma-Aldrich) chloride in the presence of riboflavin in light. One enzyme unit (U) of SOD represents the amount of enzyme causing 50% inhibition of the reduction of p-nitro-blue tetrazolium chloride. Enzyme activity was calculated in U g-1 fresh weight. CAT activity (EC 220.127.116.11) was determined by spectrophotometer based on the decomposition of H2O2, and this decrease was measured on the basis of absorbance at 240 nm. A U was determined as the amount of H2O2 (in µmol) decomposed in 1 min during this reaction. When determining POD activity (EC 18.104.22.168), the increase in absorbance was detected at 470 nm as the oxidation of guaiacol (molar extinction coefficient, ε470 = 26.6 mM-1 cm-1). The amount of enzyme that could produce 1 µmol min-1 of oxidized guaiacol was determined at 1 U.
Assay of free polyamines
The three most important APs are Put, Spd and Spm. The free PA contents were determined as described by Szepesi et al.31. Briefly, 200 mg of plants were homogenized in 5% perchloric acid. After centrifugation, the supernatant was neutralized with 2 N NaOH, then the PAs were derivatized with 10 μl of benzoyl chloride. The benzoyl-polyamines were dissolved in diethyl ether, evaporated and then separated by HPLC (JASCO, Tokyo, Japan). The standards applied were Put, Spd and Spm as hydrochlorides purchased from Merck Millipore (Darmstadt, Germany). Based on the peak areas, the results were expressed in µmol g−1 fresh weight−1.
Polyamine Catabolic Enzyme Activity Assays
Diamine oxidase (DAO, EC 22.214.171.124) and polyamine oxidase (PAO, EC 126.96.36.199) activities were estimated by spectrophotometry as described by Moschou et al.38 with some modifications. Two hundred milligrams of seedling tissue was homogenized in liquid N2, and 0.6 ml of extraction buffer was added to each sample. The extraction buffer contained 0.2 M TRIS (hydroxymethyl) aminomethane (pH 8.0), 10% glycerol, 0.25% Triton X-100, 0.5 mM phenylmethanesulfonyl fluoride (PMSF) and 0.01 mM leupeptin in 100 mM potassium phosphate buffer (pH 6.6). The homogenates were left on ice for 20 min and centrifuged for 10 min at 7000g at 4°C. Then, 150 μL of the tissue extract was combined with 0.6 mL of 100 mM potassium phosphate buffer (pH 6.6), and the reaction was started by adding 22.5 μL of 2-aminobenzaldehyde (10 mgmL−1) and 1 M Put for DAO or 1 M Spd for PAO activity measurements. After incubating the reaction mixture for 1.5 h at 37°C, the reaction was stopped by adding 50 μL of 20% (w/v) trichloroacetic acid (TCA). Absorbance was determined at 430 nm by a plate reader (Synergy HTX plate reader, BioTek Instruments, Winooski, VT, USA). Enzyme activity was expressed as specific activity (U g−1 FW), where one unit (U) represents the amount of enzyme catalyzing the formation of 1 μM Δ1-pyrroline min−1.
The data presented are the mean values of at least three independent experiments. Statistical analysis was performed with GraphPad Prism version 188.8.131.52 for Windows (GraphPad Software, La Jolla, CA, USA). Statistically significant differences were analyzed by ANOVA followed by Tukey’s post-hoc test. Results were considered significant at P
All experiments and sample collection were performed in accordance with relevant institutional, national and international guidelines and legislation. We have also obtained the corresponding authorization to collect Arabidopsis thaliana seeds and plant material.