ANALELE UNIVERSITĂŢII DIN CRAIOVA VOL. XVI (LII) – 2011 9 BIOLOGIE 9 HORTICULTURĂ 9 TEHNOLOGIA PRELUCRĂRII PRODUSELOR AGRICOLE 9 INGINERIA MEDIULUI • ANNALES OF THE UNIVERSITY OF CRAIOVA CRAIOVA – 2011 Editura UNIVERSITARIA EDITORS’BOARD: Prof. univ.dr.ing. ION VLADIMIRESCU - president Prof.univ.dr.ing. Mihail MANGRA - member Prof.univ.dr.ing. Dan POPESCU - member Prof.univ.dr.Marcel DRACEA - member Prof.univ.dr. Nicolae PANEA - member Prof.univ.dr. ing. Dumitru TOPAN - member Prof.univ.dr. Edmond-Gabriel OLTEANU - member EDITOR’S STAFF OF THE SERIES: √ BIOLOGY √ HORTICULTURE √ FOOD PRODUCE PROCESSING TECHNOLOGY √ ENVIRONMENTAL ENGINEERING Prof.dr. Popa Aurel (Romania) Prof.dr. Mitrea Ion (Romania) Prof.dr. Anton Doina (Romania) Prof.dr. Giugea Nicolae (Romania) Dr. Françoise Dosba (France) Prof.PhD. Michèle Guilloux – Bénatier (France) Prof. Jocelyne Pérard (France) Prof.dr. Balan Valerian (Republica Moldova) Prof.dr. Papachatzis Alex (Greece) Prof.dr.Marta Diszy (Spain) Prof.dr. Ştefan Nicolae (Romania) Prof.dr. Sestraş Radu Emil (Romania) Prof.dr. Hoza Dorel (Romania) Prof.dr. Grădinariu Gică (Romania) Prof.dr. Lăzureanu Aurel (Romania) Conf.dr.Sina Cosmulescu (Romania) Prof.dr. Baciu Adrian (Romania) Prof.dr. Violeta Nour (Romania) Conf.dr. Daniela Cichi (Romania) Conf.dr. Costea Dorin (Romania) Conf.dr. Mira Ionică (Romania) Conf.dr. Ciupeanu Călugăru Eleonora Daniela (Romania) ANNALES OF THE UNIVERSITY OF CRAIOVA – DOLJ A.I.Cuza Street, No. 13, Code 200585 – Craiova, Romania COMPUTERISED EDITING: Conf.univ.dr. DOINA ROŞCA Asist. univ. dr. MARIUS GRUIA I.S.S.N. 1435 – 1275 Secţiunile: HORTICULTURĂ BIOLOGIE TEHNOLOGIA PRELUCRĂRII PRODUSELOR AGRICOLE INGINERIA MEDIULUI Sections: HORTICULTURE BIOLOGY FOOD PRODUCE PROCESSING TECHNOLOGY ENVIRONMENTAL ENGINEERING Seria: 9 Biologie UNIVERSITATEA DIN CRAIOVA 9 Horticultură UNIVERSITY OF CRAIOVA 9 Tehnologia prelucrării produselor agricole 9 Ingineria mediului Vol. XVI ( LII ) - 2011 DYNAMIC CHANGES OF ANTIOXIDANT ENZYMES ACTIVITIES ON WHEAT DROUGHT TOLERANT VARIETIES DURING VEGETATION STAGES Băbeanu Cristina1, Gabriela Păunescu2 KEY WORDS: wheat, antioxidant enzymes, drought ABSTRACT The objective of this study is to investigate the behavior of five wheat cultivars in year with drought conditions. The paper presents the antioxidant enzymes activity behavior along two vegetation stages. The activity of catalase, peroxidase system and isoperoxidase, enzymes involved in the scavenging of reactive oxygen species were measured. The obtained results have showed a variation of biochemical indices analyzed both on genotype and on plant’s developmental stage. Determined biochemical indices correlated with yield data recommend selecting the tested genotypes as high productivity varieties. INTRODUCTION In Oltenia drought is becoming a major environmental constraint on crop production and one of the solicited characteristics of wheat in these area is drought tolerance (Paunescu et al. 2008). Numerous researches are carried out to obtain and select new varieties adapted to certain crop conditions, increasing the productivity, ameliorating certain characters and increasing the resistance against diseases, pathogens or drought (Mark 2007, Babeanu et al. 2010, Haris et al. 2010). Drought stress induced significant changes in normal physiological and biochemical processes. One mechanism of injury involves the breakdown of the balance between the production of reactive oxygen species and the antioxidant defense, causing accumulation of ROS which induces oxidative stress to proteins, membrane lipids and other cellular components (Nicolaeva et al. 2010). In order to limit oxidative damage under stress conditions plants have developed a series of detoxification system that break down the toxic reactive oxygen species. The plant antioxidant system is composed of both enzymatic and non-enzymatic components such as: superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), peroxidases (POX), reduced glutathione, cystein, ascorbic acid, α-tocopherol and carotenoids (Jaleel et al. 2009). With that end in view, this study aims to experiment of investigated varieties in comparative crops in the specific pedological, climatic and technological conditions of Oltenia (area characterized by a warm and drought climate) in order to point out the most stable and high performance genotypes. 1 Department of Chemistry, University of Craiova, e-mail: [email protected] 2 Research and Agricultural Development Station from Simnic 5 This paper is referring to the variation of catalase activity, peroxidase system and isoperoxidase activity in leaves of five varieties of wheat, during development time, in correlation with the proteins contents. MATERIAL AND METHOD Experiments were carried out on five varieties of Triticum aestivum (1:Dropia; 2:Boema;3:S0718; 4: S0719; 5:S0726 ) at Research and Agricultural Development Station from Simnic. These were chosen because they had a high productivity level in drought conditions. The „S.C.D.A.” geographical region Simnic is known as a drought area, with reduced rainfall and extremely high summer temperatures. These experiments were performed in highly-controlled technological condition. The plants have been complexly fertilized 12-52-0 200 kg/ha (autumn) and treated with DIVIDENT 1l/t. The soil from the investigated area is characterised by an acid pH (pH=5.7) by a low content of humus (1.8%), nitrogen (IN%=0.81), potassium (128 ppm) and by a significant supply of mobile phosphorus (54ppm). Biochemical analyses were realized on fresh leaves cut at different growing stages: phase I: tillering stage, phase II: steam elongation. Fresh tissue was homogenated with 0.1 M phosphate buffer (pH 7.0) containing 0.1 mM EDTA. Homogenates were centrifuged for 20 min at 10,000 r.p.m. and the supernatants were used for enzyme assay. The activity of ascorbate peroxidase was measures by determining the oxidative rate of ascorbic acid and expressed as μmoles ascorbic acid/1 min/1g. Total soluble peroxidase (guaiacol-type E.C.1.11.1.7) was assayed by measuring the increase in A436 due to the guaiacol oxidation and their activity was expressed as ΔA/min/1g fresh weight (Babeanu et al. 2003). Catalase (E.C.1.11.1.6) was assayed through the colorimetric method and expressed as mmoles H2O2/min/g at 25°C. The separation of soluble isoperoxidases was done by agarose gel electrophoresis at pH 8.6, performed in a horizontal gel system Statron 301 E, at 6V/cm for 4 h. Gels were stained for 30 min in 1% benzidine reagent (Babeanu et al. 2003). Quantitative evaluation of zymogrames was performed with GelAnalyzer 2010. Total soluble protein were extracted in distilled water and the concentration was evaluated by the method of Bradford using bovine albumin as a standard. RESULTS AND DISCUSSION The analyzed biochemical indices show a dependency with the investigated phase and the studied genotype. Enzyme activities and protein content in the leaves of varieties of wheat are presented in figure 1 and figure 2. The biochemical data presented confirm that the vegetation phase induces a modulation of the peroxidase system activity in plant leaves, which influences the peroxidase controlled processes. Plant peroxidases have been used as biochemical markers for various types of biotic and abiotic stresses due to their role in very important physiological processes, like control of growth by lignification, cross-linking of pectins and structural proteins in cell wall, catabolism of auxins (Selote et al. 2010). Guaiacol peroxidase, ascorbat peroxidase activity and protein content increase in the steam elongation stage, suggesting an intensification of the biosynthesis process for each studied genotype. The less sensitive among the antioxidant enzymes tested was catalase. Catalase activity was low influenced with the vegetation phase at each studied genotype suggesting that it’s activity is less important during stages when peroxidase is active, this fact confirming the hypothesis of the alternative activation and inhibition of the two hem- 6 enzymes (Shao et al. 2005). It can be observed that catalase activity didn’t significantly vary during the same developmental stages. 450 4000 300 50 400 3500 250 350 3000 40 300 g 2500 200 250 30 2000 150 200 15 0 0 20 15 0 100 10 0 10 0 0 50 10 50 500 A/min/1g A/min/1g moles/min/g μ Δ 0 0 0 0 catalase activity Dropia Boema S0718 S0719 S0726 m m oles H 2O 2/m in/ Dropia Boema S0718 S0719 S0726 ascorbat peroxidase Peroxidase activity activity Peroxidase protein mg/g protein Peroxidase act. I Peroxidase act. II protein I protein II catalase act.I catalase act. II APO I APO II Figure 1. Peroxidase, catalase activity Figure 2. Ascorbat peroxidase, protein content In the cases of all studied genotypes, three anionic isoperoxidase fractions and a cationic one in the first vegetation stage can be observed; and two anionic isoperoxidase fractions and one cationic fraction for the second vegetation stage. The quantitative evaluation shows the isoperoxidase activity depends on the genotype, as well as on the vegetation stage (figure 3). The isoperoxidase activity shows a considerable increase in case of Dropia and Boema genotypes at steam elongation. For the S0718, S0719 and S0726 the isoperoxidasic activity slightly increased at second vegetation stage. The obtained electrophoregrames are shown in figure 4 Isoperoxidase pattern /phase I Isoperoxidase pattern phase II 3500 3500 3000 3000 2500 2500 2000 2000 1500 1500 1000 1000 500 500 0 0 Dropia