Extraction and purification of defensin from Trigonella foenum-graecum seeds
The natural defensin from 50-g powdered Fenugreek (Trigonella foenum-graecum) seeds, FD, was extracted and purified, according to Oddepally and Guruprasad (2015) as follows: Fine flour (100 g) was prepared from the seeds of Fenugreek in a mill. A protein extract was prepared from this flour, using 500 ml of extraction buffer (10 mM Na2HPO4, 15 mM NaH2PO4, 100 mM KCl, 1.5% EDTA, pH 5.4) for 2 h at 4 °C with constant agitation. This protein extract was centrifuged at 15,000g, and the supernatant was fractionated at 70% relative ammonium sulfate saturation at 4 °C for 18 h. After centrifugation under the same conditions, the precipitate was re-dissolved in distilled water and heated at 80 °C for 15 min in a water bath. This heated protein extract was centrifuged at 3000 rpm for 5 min. The supernatant was recovered and extensively dialyzed against distilled water for 3 days and then recovered by freeze-drying. For peptides, purification was initially performed on a DEAE-Sepharose column (with 100 ml of resin), equilibrated with 20 mM Tris–HCl (pH 8.0), at a flow rate of 60 ml/h. The freeze-dried protein extract (50 mg) was reconstituted in 5 ml of the equilibrium buffer and centrifuged (6000 rpm, 3 min at 4 °C), and the supernatant was loaded onto the column. A non-retained fraction (D1) was eluted in the equilibrium buffer and used as defensin source. The protein content in the purified extract and the crude extract was determined by Lowry et al. (1951) method and subjected to protein electrophoresis.
Culturing of plant pathogenic fungus
Fusarium oxysporum was friendly obtained from Assistant Professor: Manal Tawfik, Assistant Professor of Mycology, Botany Department, Faculty of Science, Zagazig University, Egypt.
The fungus spores were prepared by growing the organism on Hordeium grains medium. Five hundred-milliliter flasks, containing 100-g washed Hordeium grains and 80-mL tap water, were autoclaved at 121 ± 1 °C for 15 min. The autoclaved Hordeium grains were inoculated with the fungus under aseptic conditions and incubated at 25 ± 1 °C for 21 days. The medium was mixed and rubbed together to release mycelium and spores from Hordeium grains. The mycelium and spores were taken in sterile water and used for inoculation of plants. Calculate spores concentration using a hemocytometer. Prepare 106 spores/ml suspensions in autoclaved distilled water for inoculation (Hegazy 2008).
Pot experiment
Wheat (Triticum aestvium L.; cultivar Sakha 96) grains were obtained from the Agricultural Research Center, Giza, Egypt.
Pot experiments were carried out in the greenhouse of the Botany Department, Faculty of Science, Zagazig University, Egypt. Plastic pots (20-cm diameters) were filled with about 2 kg of soil (peat moss soil) each. The pots were divided into two groups. The first remained as it is, while the second was infected by 100 ml of the prepared F. oxysporum spore suspension.
Grains were surface sterilized by 0.1% mercuric chloride solution followed by rinsing with distilled water. A group of the seeds was soaked in the purified extracted defensin solution for 12 h then dried again to get rid of excess moisture (defensin-primed group), and the other group was kept dry (non-primed group). Each group was planted in the plastic pots (seven grains in each pot). Pots were arranged on the greenhouse benches and kept under natural photoperiod (12 to 13 h), temperature (28 ± 4 °C) and irrigated weekly. Samples were collected after 10 days (1st stage) and 20 days (2nd stage). The experiment was done in triplicate.
Markers of oxidative stress
H2O2 content in 1-g fresh leaves was determined according to the method of Alexieva et al. (2001).
MDA (malonyldialdehyde) determination in 1-g fresh leaves has followed the method described by Li (2000).
Antioxidant activity
Five-gram fresh leaves were homogenized in 0.05 M cold phosphate buffer (pH 6.5) containing 1 mM EDTA, Na2 and centrifuged at 3800g for 10 min. The supernatant was completed to a total known volume (5 ml) and used as the protein and enzyme source. The residue was carefully washed with distilled water and centrifuged several times. The wall fraction was then kept with 10 ml of 1 M NaCl for 1 h to release cell wall-bound POX and centrifuged at 3800g for 10 min; the supernatant was used as the source of wall-bound POX (Saroop et al. 2002).
SOD activity was measured by the nitro blue tetrazolium (NBT) reduction method (Beyer and Fridovich 1987). PPO was estimated according to Kar and Mishra (1976). Soluble and cell wall-bound (POX) was determined according to Saroop et al. (2002). The enzymes’ activity was expressed as U mg−1 protein min−1.
Free and cell wall-bound phenols were extracted from 1-g leaves of both control and treated plants according to the method of Campbell and Ellis (1992).
Proline content was determined in 1 g of Fresh leaves according to the method of Bates et al. (1973).
Determination of hydrolysis processes
Carbohydrates hydrolysis
The amylase activity was measured according to Johnson (2007) and expressed as the amount of starch hydrolyzed min−1 mg protein−1.
Carbohydrates were estimated in 1 g of fresh leaves according to phenol-sulfuric acid method (Dubois et al. 1956).
Organic phosphorus hydrolysis
The activities of the two enzymes were assayed depending on the method of Tominaga and Takeshi (1974). One nkat of enzyme activity was defined as 1 nmol p-nitrophenol liberated min−1 and specific activity as nkat mg−1 protein.
For determination of the total phosphorus content, 5 g of dried powder of plants was digested in a mixture of concentrated nitric acid, sulfuric acid, and perchloric acid at the ratios 10:1:4, respectively. The volume was made up to a constant volume with distilled water according to the method of Chapman and Pratt (1978). Phosphorus content in the digested samples was determined according to Murphy and Riley (1958). Results were expressed as milligrams per gram dry weight.
Protein hydrolysis
Protease activity was measured in an azocasein assay (Mel et al. 2000). Specific enzyme activity was expressed as change in optical density mg−1 protein min−1.
The total protein content was identified according to the method of Lowry et al. (1951).
Total free amino acids were estimated in 1 g of fresh leaves according to the method of Lee and Takahashi (1965).
Metabolic activity marker (shikimic acid) content
Shikimic acid concentration in 1 g of fresh leaves was determined according to Zelya et al. (2011). The frozen plant leaves or calli were ground (0.2 g) before 1 ml/100 mg biomass of 0.25 M HCl was added. The extracts were shaken (2 min.) and then centrifuged at 3800g for 30 min. The supernatant (50 μl) reacted with 0.5 mL of a 1% solution of periodic acid. After 3 h at room temperature, 0.5 ml of 1 M sodium hydroxide and 0.3 ml of 0.1 M glycine were added per sample. Samples were centrifuged again and absorbance measured at 380 nm. The amount of shikimic acid in the test sample was calculated using the standard curve.
Hormones
The method of hormone extraction was essentially similar to that adopted by Shindy and Smith (1975) and described by Hashem (2006). To estimate the amounts of acidic hormones abscisic acid (ABA), the plant hormone fractions and standard ones were methylated according to Vogel (1975) to be ready for gas chromatography (GC) analysis. Flame ionization detector was used for the identification and determination of acidic hormones using Hewlett Packard Gas Chromatography (5890) fitted and equipped with HP-130 mx 0.32 mm × 0.25 mm capillary column coated with methyl silicone. The column oven temperature was programmed at 10 °C min−1 from 200 °C (5 min) to 260 °C and kept finally to 10 min. Injector and detector temperature were 260 and 300 °C, respectively. Gas flow rates were 30, 30, and 300 cm s−1 for N2, H2, and air, respectively, and flow rate inside column was adjusted to 2 ml min−1. Jasmonic acid (JA) was determined, according to Kramell et al. (1997) using NUCLEODEX beta-PM, 200 mm and 4 mm ID column, flow rate adjusted at 1 ml min−1 and detected at UV 210 nm. Standards of ABA and JA were used. Peak identification was performed by comparing the relative retention time of each peak with those of ABA and JA standards. Peak area was measured by triangulation, and the relative properties of the individual components were therefore obtained at various retention times.
Statistical analysis
All results were analyzed by SPSS software (version 14). Data was expressed as mean ± SD. Comparison of mean values of the sample and the control was done, using paired T test. P < 0.05 was considered to be significant (Levesque 2007).