Methylene blue increases the tolerance of tomato plants to abiotic stresses By B. ALONI*, L. KARNI and H. AKTAS 1 Department of Vegetable Research, Institute of Plant Science,ARO,The Volcani Center, P.O. Box 6, Bet-Dagan 50250, Israel (e-mail: vcaloni@volcani.agri.gov.il) (Accepted 13 May 2010) SUMMARY Environmental stresses such as high temperature and high salinity are known to cause oxidative stress in plants by enhancing the generation of reactive oxygen species (ROS), which have a strong impact on plant development. Mitochondria constitute one of the major sources of ROS in roots. Recently, it has been shown in mammalian systems that methylene blue (MB) at very low concentrations can attenuate mitochondrial ageing by scavenging ROS. In the present study, we tested whether MB could be used to protect tomato (Lycopersicon esculentum L.) seedlings against the harmful effects of high salinity and high root temperature.Tomato seedlings were grown hydroponically and were subjected to two abiotic stresses: high salinity (150 mM NaCl) in the nutrient medium, or high root temperature (35ºC) for 14 d. These stress treatments were applied with or without the addition of 10 –8 M MB. The results showed that, under normal conditions, MB enhanced root and shoot growth, increased root respiration, decreased root H 2 O 2 and malonyldialdehyde (MDA) concentrations, and enhanced root superoxide dismutase (SOD) activity. Both high salinity and high root temperature stress impaired root and shoot growth, and caused an enhancement of root oxidative stress. MB had a significant protective effect against both abiotic stresses and restored the levels of oxidative stress-related components (i.e., H 2 O 2 and MDA) in root and leaf tissues almost to normal levels. It is hypothesised that MB could confer its protective effect not only through interactions with root mitochondria, but also via additional cellular targets in the root. H igh temperature and high salinity are among the most important environmental factors that limit the growth and productivity of crop plants. In many areas, soil temperatures often reach high values during the Summer, which has a strong influence on shoot and root growth, and on the survival of whole plants (Paulson, 1994). Soil and water salinity often reach levels that also impair crop production (Kafkafi and Bernstein, 1996). Soil temperatures in the range of 35º – 40ºC can inhibit root growth, as well as both mineral and water uptake, partly by hampering the respiratory machinery. Rachmilevitch et al. (2006) demonstrated that the thermo-tolerance of roots was dependent on their respiratory efficiency and, in addition, it is well-documented that stresses such as extreme temperature, high light intensity, water deficit, and/or high salinity, cause oxidative stress in plants (Aktas et al., 2005; Burdon et al., 1996; Gossett et al., 1994; Hernandez et al., 2000; Mitler, 2002). Although reactive oxygen species (ROS) play central roles in root functions such as elongation (Ivanov, 2007), branching (Pasternak et al., 2005), gravitropism, and mineral uptake (Shin and Schachtman, 2004), and in stress responses (Mitler, 2002), when ROS reach high levels they may be toxic to plant tissues. Karni et al. (2009) found that roots accumulated malonyldialdehyde (MDA), a product of fatty acids oxidation, and H 2 O 2 , (a toxic radical) under growth- inhibiting levels of salinity. One of the main sources of oxygen free radicals, especially in non-photosynthetic plant organs, are the mitochondria (mtROS; Puntarulo et al., 1988; Rhoads et al., 2006). The known sites of mtROS production are complex I and complex III of the electron transport complex (ETC), where superoxide radicals (O 2 .– ) are formed and rapidly dismutated to H 2 O 2 , which is also toxic at high concentrations. These toxic radicals may damage mitochondrial components and functions (Almeras et al., 2003; Sweetlove et al., 2002; Kristensen et al., 2004; Tiwari et al., 2002). Recently, it has been shown that mitochondrial activity in roots has an important role in root development. This was shown using transgenic tomato plants in which the expression of either mitochondrial malate dehydrogenase, or fumarase, was impaired. This resulted in substantial reductions in the deposition of root dry matter (DM) and in respiratory activity (van de Merwe et al., 2009). Methylene blue (MB) is a heterocyclic aromatic molecule that is widely used as a redox indicator in analytical chemistry and as a dye for staining DNA and/or RNA. It has been in human clinical use to treat a variety of ailments for approx. 100 years (Pelgrims et al., 2000; Kwok and Howes, 2006; Patel, 2006). Recently, MB was also shown to delay the senescence of mammalian cells by increasing mitochondrial respiration (Atamna et al., 2008). It was suggested that MB could affect mitochondrial longevity through its role in the reduction- oxidation cycles of mitochondrial electron transport (see Atamna et al., 2008; and references, therein). In the present research, we postulated that, through its role in reducing oxidative damage in the mitochondrial respiratory chain, MB might have a beneficial effect on plant responses to abiotic stress, and thereby increase plant *Author for correspondence. 1 Present address: Suleyman Demirel University, Agriculture Faculty, Horticulture Department, East Campus, 32260 Isparta, Turkey. Journal of Horticultural Science & Biotechnology (2010) 85 (5) 387–393