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ENHANCED EXPRESSION AND ACTIVATION OF THE ALTERNATIVE OXIDASE DURING OZONE TREATMENT IN TOBACCO PLANTS Luisa Ederli (1), Nicola Tosti (2) and Stefania Pasqualini(1) (1) Dip. di Biologia Vegetale e Biotecnologie Agroambientali, UNI Perugina ([email protected]) (2) Istituto di Genetica Vegetale - CNR Perugia In addition to the cytochrome respiratory pathway, plants display an alternative pathway that branches from the main respiratory chain at the level of ubiquinone pool (Vanlerberghe et al., 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48: 703-734); it is not inhibited by cyanide and does not contribute to the formation of a proton gradient. This pathway is therefore not coupled to ATP synthesis (Siedow and Umbach, 1999, Plant Cell, 7: 821-831). Electron transfer along this alternative pathway involves only a single protein, termed alternative oxidase (AOX), which catalyses the four-electron reduction of oxygen to water. The physiological role of this pathway remains unclear except in the specialized case of promoting thermogenesis during flowering in aroid spadices (Moore and Siedow, 1991, Biochim. Biophys. Acta, 1058: 121-140). However, it has been proposed that AOX can function as a mechanism to decrease the formation of ROI (reactive oxygen intermediate) under different stress conditions (Hiser and McIntosh, 1990, Plant Physiol., 93: 312-318; Vanlerberghe and McIntosh, 1992, Plant Physiol., 100: 115-119; Lennon et al., 1997, Plant Physiol., 115: 783-791). In this work we report the effect of an acute ozone fumigation (150 ppb for 5 h) on cyanide-resistant respiration in Bel W3 tobacco plants. A strong induction of expression of alternative oxidase was documented 2.5 h after the onset of fumigation. The increase in mRNA correlated with the increase in AOX protein. Oxygen electrode measurements in isolated leaf mitochondria revealed that 2.5 h after the onset of fumigation there was a strong enhancement of alternative oxidase capacity, whereas the capacity of cytochrome respiratory pathway was significantly reduced. Western blot analysis showed, at the same time, a decrease of mitochondrial cytochrome c. The results indicate that O3-induced oxidative stress stimulates AOX in Bel W3 plants as a strategy to keep low mitochondrial ROI formation thus limiting cell death.