purification and properties of lipoxygenase from wheat seedlings infected by Fusarium graminearum and treated by salicylic acid

Lipoxygenase from wheat seedlings in normal conditions, infected by Fusarium graminearum and treated by salicylic acid was isolated. The isolated enzyme was purified by the methods of salting-out (60% ammonium sulphate), dialysis, gel-filtration and ion-exchange chromatography. Specific activity of the purified enzyme was 8.0-12.5 ΔЕ234/mg of protein, degree of purification – 11.6-15.3 times. The enzyme yield was 18.3-27.9%. Molecular mass of lipoxygenase is 90 kDa, amino acid composition is distinguished by a high content of glutamic acid, proline, valine, isoleucine, leucine and low level of histidine, tyrosine, phenylalanine, threonine, tryptophan, cystein. Research of lipoxygenase substrate dependence indicated that the enzyme catalysed with the maximum velocity of the reaction of arachidonic acid oxidation at a substrate concentration of 4.5 mM at pH 7.2, the reaction of linoleic acid oxidation at a substrate concentration of 4.5 mM at pH 7.2 and the reaction of linolenic acid oxidation at a substrate concentration of 9.0 mM at pH 8.0. The change of wheat lipoxygenase activity depending on genotype resistance to Fusarium graminearum and millieu of germination was shown. One of the manifestations of the protective effect of salicylic acid is its ability to induce changes of lipoxygenase activity.


e x p e r i m e n t a l w o r k s e x p e r i m e n t a l w o r k s
Plant Breeding and Genetics institute-national center of seed and cultivar investigation, ukraine; e-mail: olgamolod@ukr.net; 2 Оdessa national medical university, ukraine; e-mail: 93vi_63@mail.rulipoxygenase from wheat seedlings in normal conditions, infected by Fusarium graminearum and treated by salicylic acid was isolated.The isolated enzyme was purified by the methods of salting-out (60% ammonium sulphate), dialysis, gel-filtration and ion-exchange chromatography.specific activity of the purified enzyme was 8.0-12.5 ΔЕ 234 /mg of protein, degree of purification -11.6-15.3times.The enzyme yield was 18.3-27.9%.molecular mass of lipoxygenase is 90 kda, amino acid composition is distinguished by a high content of glutamic acid, proline,valine, isoleucine, leucine and low level of histidine, tyrosine, phenylalanine, threonine, tryptophan, cystein.research of lipoxygenase substrate dependence indicated that the enzyme catalysed with the maximum velocity of the reaction of arachidonic acid oxidation at a substrate concentration of 4.5 mm at ph 7.2, the reaction of linoleic acid oxidation at a substrate concentration of 4.5 mm at ph 7.2 and the reaction of linolenic acid oxidation at a substrate concentration of 9.0 mm at ph 8.0.The change of wheat lipoxygenase activity depending on genotype resistance to Fusarium graminearum and millieu of germination was shown.one of the manifestations of the protective effect of salicylic acid is its ability to induce changes of lipoxygenase activity.k e y w o r d s: Triticum aestivum l., lipoxygenase, purification and properties, fusariosis, salicylic acid.I t is known that enzymes participating in lipid metabolism may take an active part in forming defense responses of plants.Activation of lipa ses and increase of free fatty acids, which can further be metabolized through lipoxygenase path way, occur as a results of interation between elici tors and receptors of plasmalemma.Lipoxygenase (EC 1.13.11.12,LOG) is one of the key enzymes of lipoxygenase metabolism that results in formation of compounds, being toxic for most pathogens, as well as phytodienic and jasmonic acids.Interest to the given enzyme is also caused by the fact that cataly zing the process of peroxide oxidation of unsaturated fatty acids, lipoxygenase can affect the composition of membrane lipids and functions of plant cell bio membranes [4,5].
The process of pathogens identification in plants proceeds with the help of signal systems which determine the response of cells to various chemicall and physical effects.The number of com pounds, performing the function of the signal system mediators, constantly grows.Salicylic acid belongs to such compounds.It takes part in activation of the whole complex of protective responses of plants that are important for forming resistance to biotic and abiotic stresses [6].
In spite of broad distribution and important functions of lipoxygenase in the plant and animal organisms, the structure, activation mechanisms and role of this enzyme in forming the mechanisms of plants resistance, wheat in particular, is not suffi ciently studied.Thus, the isolation and investigation https://doi.org/10.15407/ubj88.06.026 of lipoxygenase from the wheat plants distinguished by the level of resistance to fusariosis, one of the most harmful diseases, and grown on the media con taining pathogenic infection and solution of salicylic acid, the inducer of plant protective reactions, is ur gent and important for finding out the enzyme struc ture and its role in defense mechanisms of the wheat plants.In this connection the objective of this study consisted in performing the isolation, purification and characterization of properties of lipoxygenase from the wheat seedlings grown in normal condi tions, infected by the fusariosis agents and treated by the inducer of resistance (salicylic acis (SA)).

materials and methods
Lipoxygenase was isolated from the over ground part of 4day seedlings of the wheat (Triticum aestivum L.) genotypes, distinguished by re sistance to fusariosis agents (line Erythrospermum 84/06 -resistant to fusariosis; variety Odeskaya polukarlikovaya (Odessa halhdwarf) -suscepti ble to fusariosis.A highly pathogenic strain K90 Fusarium graminea rum served as the infection agent.Colonies of Fusarium graminearum K90 from the collection of strains of the Department of Phytopathology and Entomology of Plant Breeding and Genetics Institute -National Center of Seed and Cultivar Investigation were grown on the potato agar.To obtain filtrate the pore suspension was in troduced into the liquid potato medium and culti vated for 10 days at 24 °С.The undamaged wheat cariopses grown on filter paper in thermostate at 25 °С and relative air humidity 60% during 4 days were used in the experiments.The infection of plants with the pathogen was conducted, when growing the seedlings in the medium which contained 1×10 5 conidia/ml of the pathogenic strain K90 Fusarium graminearum.Treatment of plants with salicylic acid was conduc ted when growing the plants for 4 days on the filter paper wetted with 2 mM solution of sali cylic acid.Under mutual action of the pathogen and salicylic acid the grains moistened during a night in the 0.1 mM solution of salicylic acid were set out on suspension with the pathogen.The exposure being over, the prepared overground part of sprouts was frozen at -70 °С, liophylically dried and ground.
The stages of isolation and purification of lipo xygenase from the overground part of seedlings con sisted of the extraction by 0.05 M phosphate buffer , pH 7.2, salting out by 60% ammonium sulfate, dia lysis, Sephadex G100 gelfiltration (Pharmacia, Sweden) and Sepharose CL6B ionexchange chro matography (Pharmacia, Sweden).Column chroma tography of lipoxygenase was performed with the help of the equipment of the firm LKB (Sweden), using a lowtemperature cabinet (Combicoldrac II), peristaltic pump (Microperpex), ultraviolet detec tor with a flowtype cuvette (Uvicord S), collec tor of fractions (Ultrorac II), columns 2.6×60 cm, 1.6×95 cm (for G100), 1.6×70 cm (for DEАE Sepharose CL6B) at 4 °С.Treatment of the Sepha dex G100, ion exchanger DEAESepharose CL6B was performed following standard methods [7,8].The enzyme activity was determined spectrophoto metrically by the reaction of fermentative formation of fatty acid hydroperoxides, recorded at 234 nm.A mixture of fatty acids isolated from walnut kernels was used as a substrate.The content of linoleic acid in the mixture was 67%.Concentration of the acid in the mixture was 2.8 mM.The extinction change by 0.01 for 1 min was taken as the unit of lipoxygenase activity.The total protein content in the extract was determined by the Lowry method [10].The isolated lipoxygenase was determined by the method of ana lytical electrophoresis in PAAG in the alkali buffer system at pH 8.3, using a specific oxidation reaction of linoleic acid in the presence of starch and potas sium iodide [11,12].To reveal the zone of lipoxy genase location in gels we used the method by Heil with coauthors [13], based on formation of stained iodinestarch complex in the presence of potassium iodide and linoleic acid hydroperoxides.This in mind soluble starch was introduced before polym erization in gel to concentration of 1%; electropho resis being finished, gel was incubated for 30min in 0.51.0%solution of sodium salt of linoleic acid.Then the gel, thoroughly washed in distilled water, was placed into mixturedevelo per which consisted of 100 ml of 7% acetic acid and 5 ml of freshly pre pared saturated solution of KI.The appearance of brownviolet stripes on the yellow background of the gel evidenced for availability of lipoxygenase isoforms.
Amino acid analysis of lipoxygenase was per formed by automatic analyzer of amino acids Hi tachi835 (Japan).The samples were hydrolyzed in 6 M hydrochloric acid at 105 °С in hermetically sealed flasks during 24 h.After hydrolysis the sam ples were stewed in the rotor evaporator Rodatest (Hungary), and the precipitate was dissolved in 0.02 M hydrochloric acid.
Statistical processing of the investigation re sults was made using the pack of programs Analy sis of the Data of Electron Tables Microsoft Excel, ima ge analysis programs Imagel, AnaIS.The experi ments were performed in triple biological and ana lytical repetitions.Mean values and their standard errors are presented in Fig. 5 and Table 2, differences between the experiment variants, distinquished by genotypes resistance to parthogen, were considered reliable at the significance level P < 0.05 by the Stu dent criterion.

results and discussion
Lipoxygenase was extracted from the seedlings tissues by 0.05 M phosphate buffer, pH 7.2 at 4 °С during 30 min at the ratio mass:volume 1 : 10.Ho mogenate was centrifuged during 20 min at 6 000 g (4 °С), using a supernatant for further purification.Our purification plan includes 4 stages.
stage 2. dialysis.The sediment was dissolved by 0.05 M phosphate buffer at pH 7.2, and put to dialy sis against 20fold volume of distilled wa ter during night at 4 °С, using dialysis membranes (Regene rated cellulose tubular membrane, Fisher Scientific, USA) with pore diameter of 1214 kDa.After dialysis the enzyme solution was centrifuged for 20 min at 6 000 g (4 °С), the enzyme activity and protein content were determined in supernatant.The latter was used for further purification.stage 3. Gel-filtration.The enzyme solution was lyophilically dried, dissolved in 0.05 M phos phate buffer at pH 7.2, and gelfiltration was per formed on the column (2.6×60 cm) with Sephadex G100 in the same buffer with velocity 7.5 ml/cm 2 •h, gathering fractions of 5 ml.The fractions with maxi mum enzyme activity were united and used for fur ther purification.stage 4. ion-exchange chromatography.The enzyme solution was applied to the column (1.6×70 cm) with DEAESepharose CL6B, balan ced with the above buffer solution.Lipoxygenase was eluted with linear gradient NaCl from 0.01 to 0.5 М with flow velocity 3 ml/cm 2 •h, gathering frac tions of 5 ml.The fractions with maximum enzyme activity were extracted.
After the extraction, salting out by ammonium sulfate with following dialysis the specific activity of lipoxygenase from the wheat seedlings was 0.288 0.530 exp.un/min/mg of protein.
Sephadex G100 gelfiltration has helped to purify lipoxygenase from considerable amount of lowmolecular impurities of nonprotein nature.Li poxygenase yield from the wheat seedlings in the region of proteins with molecular mass of 97 kDa, 67 kDa, and 20 kDa (Fig. 1) had 3 peaks.After gel filtration the enzyme specific activity increased 1.532.53times.Fractions obtained after gelfiltra tion and characterized by the enzyme maximum activity (peaks 12), were lyophilized and applied to the column with DEAESepharose CL6B.Un der DEAESepharose CL6B chromatography the lipoxygenase activity was found in the form of two peaks (Fig. 2).The first peak appeared at NaCl con centration equal to 0.05 M, while the second peak appeared under the gradient 0.050.2M NaCl.Maxi mum activity of the enzyme was found in the first peak.The material, which activity was 18.327.9times higher compared with lipoxygenase activity after salting out, was obtained as a result of using the DEAESepharose CL6B chromatography.As a result of using Sephadex G100 gel fil tration and DEAESepharose CL6B ion exchange chromatography the yield of lipoxygenase prepara tion from the control samples was 37.7% with pu rification coefficient 27.7; from seedlings treated with SA -42.7% with purification coefficient 18.32; from infected speedlings 56.6% with purification coefficient 27.95% and from infected seedlings after preliminary treatment of grain by SA 48.8% with purification coefficient 27 (Table 1).Molecular mass of isolated enzymes, determined by the method of Sephadex G100 gelfiltration, was 90 kDa (Fig. 3).Results of electrophoretic analysis of isolated en zymes are presented on Fig. 4.
When determining enzyme activity in condi tions of the incubation medium pH 4.510.0, it was found that its optimum for lipoxygenase of all the studied samples is within 7.88.0.The investigation of substrate dependence of lipoxygenase activity in the peroxidation reaction of linoleic, linolenic and arachidonic acids has shown the effect of more intensive oxidation of arachidonic acid under the substrate concentration 4.5 mM with pH 7.2, linoleic acid under substrate concentration 4.5 mM with рН 7.2 and linolenic acid under sub strate concentration 9.0 mM with рН 8.0 (Table 2).
One of possible functions of lipoxygenase is its participation in formation of plant protection reac tions against pathogenesis.To confirm this suppo sition we have studied lipoxygenase activity in the control plants, those infected by Fusarium graminearum, treated by SA in wheat genotypes distinguished by the level of resistance to fusariosis agents.The conducted studies have shown that the 2.3 and 2.6 fold increase of enzyme activity compared with control, respectively, was observed in the resistant wheat genotype under the effect of pathogen and SA.
The enzyme activity of a susceptible genome did not practically differ from the control plants under seed lings infection by Fusarium graminearum and in considerably but reliably increased at Р ≤ 0.05 under the effect of SA (Fig. 5).The enzyme activity in the stable genotype seedlings, grown on the pathogen suspension after preliminary grain treatment with SA, reliably increased in respect of the control, but the enzyme activation level was lower than that in the infected seedlings treated with SA.Lipoxyge nase activity in the seedlings of susceptible genotype under joint action of pathogene and SA, as well as in the infected plants treated with SA, increased in considerably but reliably at Р ≤ 0.05 in respect of the control and infected seedlings.A similar response to the joint action of fusariosis infection and SA was established by the change of trypsin activity inhibi tors [15].
Such a response of the wheat plants to the ac tion of salicylic acid and fusariosis infection evi dences that the joint action of these factors causes complex interaction directed to preservation and increase of plants resistance to the given pathogen.The wheat plants response to fusariosis agent infec tion and the effect of salicylic acid is regulated by the interaction of separate components of biochemical defense system.The regulating effect of biochemical plants is determined by the effect factor nature, mo bilization velocity of the defense mechanisms which have their display peculiarities depending on the level of the genotype stability.The results obtained evidence for lipoxygenase involvement in the regula tion of cell metabolism of the wheat plants under the effect of fusariosis infection and salicylic acid.