Analele Ştiinţifice ale Universităţii „Alexandru Ioan Cuza”, Secţiunea Genetică şi Biologie Moleculară, TOM X, 2009 RESPONSE OF BARLEY SEEDLINGS TO OXIDATIVE STRESS GENERATED BY TREATMENTS WITH GROWTH HORMONES ZENOVIA OLTEANU 1* , LĂCRĂMIOARA OPRICĂ 1 , ELENA TRUŢĂ 2 , MARIA MAGDALENA ZAMFIRACHE 1 Key words: growth hormones, Hordeum vulgare cv. Madalin, oxidative stress, protein content Abstract: The effects induced by growth hormone regulators on soluble protein level and some oxidoreductases in Hordeum vulgare cv. Madalin seedlings were investigated. The study of superoxide dismutase, catalyse and peroxidase behaviour and of protein synthesis was realized in dynamics to evaluate the response of barley seedlings to oxidative stress generated by exposure to hormone factors. During experiments, peroxidase registered smaller limits of variability than superoxide dismutase and catalase. It seems that superoxide dismutase and catalase rather than peroxidase acted as components of an antioxidative protective system. Generally, the protein level not presented significant modifications in early stage, but at 6 days, the major part of variants responded by amplification of protein synthesis, excepting some kinetin and GA3 concentrations. In final stage, both control and hormone variants reduced their protein synthesis. INTRODUCTION Phytohormones have essentials roles in the regulation of plant physiological processes, because they serve as integrators and inductors of multicellular plant organism differentiation and in expression of hereditary information. The organ growth and development depend on endogenous hormone level. Various factors such as organism physiological state, correlation degree between characters, ontogenetic phase, gene epistasy or gene pleiotropy, and environmental conditions can induce quantitative modifications of endogenous phytohormones (DUCA et al., 2003). Plants are very often subjected to a wide variety of stressful conditions (UV-radiation, high light intensities, exposure to herbicides, extreme temperatures, toxins, air pollutants, heavy metals, wounding etc.) that generate reactive oxygen species (ROS) and create oxidative stress situations. Oxidative stress can arise from an imbalance between generation and elimination of ROS, leading to excess ROS levels which can damage all biomolecules and can lead in turn to various diseases and cell death. Increase of endogenous ROS levels and activation of antioxidant defence mechanisms are the most rapid indicators of oxidative stress. ROS (superoxide anion radical, singlet oxygen, hydrogen peroxide, hydroxyl radical) can damage many cellular components including proteins, membrane lipids, nucleic acids. A severe effect is peroxidation of membranary lipids (GONÇALVES et al., 2007). To minimize the damaging effects of ROS, aerobic organisms evolved non-enzymatic (ascorbic acid, reduced glutathione, carotenoids, tocopherols, flavonoids, alkaloids) and enzymatic (superoxide dismutase, SOD; catalase, CAT; peroxidase, POD) antioxidative protection mechanisms. SOD - the main scavenger of superoxide radicals - is a strong antioxidant which converts the toxic superoxide in hydrogen peroxide and oxygen by so called dismutation reaction: 2O 2 - + 2H + → H 2 O 2 + O 2 . The formed H 2 O 2 in this way is also toxic and must be scavenged by catalase and diverse peroxidases which catalyze its conversion to H 2 O. These enzymes that interact with superoxide and H 2 O 2 are tightly regulated through a feedback system. For example, excessive superoxide inhibits POD (mainly glutathione peroxidase) and CAT to modulate the equation from H 2 O 2 to H 2 O; increase of H 2 O 2 slowly inactivates SOD. Therefore, CAT and glutathione peroxidase, by reducing H 2 O 2 , conserve SOD; SOD, by reducing superoxide, conserves catalases and glutathione peroxidase. Through this feedback system, steady low levels of SOD, glutathione peroxidase, and catalase, as well as superoxide and H 2 O 2 are maintained, which keeps the entire system in a fully functioning state (SCANDALIOS, 2005). Depending on H 2 O 2 concentration, CAT exerts a dual function. At low H 2 O 2 levels (<1 ȝM) and in presence of increased levels of other substrata (ethanol, ascorbic acid etc.), CAT acts like a POD: RH 2 + H 2 O 2 → R + 2H 2 O. At high H 2 O 2 concentrations, CAT degrades extremely rapid H 2 O 2 by catalasic specific reaction: 2 H 2 O 2 → 2H 2 O + O 2 . POD catalyzes the oxidation of many substrata (phenols, aromatic amines, ascorbic acid, glutathione, nitrites) in the presence of H 2 O 2 , with H 2 O production: AH 2 + H 2 O 2 → A + 2H 2 O. Peroxidase is present both in cytosol and in cell wall, in genetically different isoenzyme forms (GASPAR et al., 1982; SCANDALIOS, 2005). MATERIAL AND METHODS Biological material consisted in Hordeum vulgare L. cv. Mădălin caryopses, from Podu Iloaie Agricultural Research Center. They were subjected to 4 hours treatments, by immersion in growth regulators solutions: 2,4-D (A), kinetin (B), gibberellic acid (C), 2,4-D + Kin mixture (D), as follows: Control distilled water A1 1 mg l -1 2,4-D A2 10 mg l -1 2,4 D A3 25 mg l -1 2,4-D A A4 50 mg l -1 2,4-D B B1 1 mg l -1 kinetin 29