Original article Sensitivities of the alternative respiratory components of potato tuber mitochondria to thiol reagents and Ca 2+ André B. Mariano, Caroline Valente, Silvia M.S.C. Cadena, Maria E.M. Rocha, Maria B.M. de Oliveira, Eva G.S. Carnieri * Laboratório de Oxidações Biológicas, Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, 81.531-990 Curitiba, PR, Brazil Received 12 July 2004; accepted 14 December 2004 Available online 01 February 2005 Abstract Plant mitochondria differ from those of mammals, since they incorporate an alternative electron transport pathway, which branches at ubiquinol to an alternative oxidase (AOX), characteristically inhibited by salicylhydroxamic acid (SHAM). Another feature of plant mito- chondria is that besides complex I (EC 1.6.5.3) they possess alternative NAD(P)H-dehydrogenases insensitive to rotenone. Many stress conditions are known to alter the expression of the alternative electron transport pathway in plant mitochondria. In the present study we investigated the effects of some thiol reagents and Ca 2+ on potato mitochondrial respiratory chain presenting different activities of the alter- native respiratory componentsAOX and external NADH dehydrogenase, a condition induced by previous treatment of potato tubers (Solanum tuberosum L., cv. Bintje) to cold stress. The results showed that Ca 2+ presented an inhibitory effect on AOX pathway in potato mitochondria energized with NADH or succinate, which was only now observed when the cytochrome pathway was inhibited by cyanide. When the cytochrome pathway was functional, Ca 2+ stimulated the external NADH dehydrogenase. Diamide was a potentAOX inhibitor and this effect was only now observed when the cytochrome pathway was inactive, as was the case for the calcium ion. Mersalyl inhibited the externally located NADH dehydrogenase and had no effect on AOX activity. The results may represent an important function of Ca 2+ on the alternative mitochondrial enzymes NADH-DH ext and AOX. © 2005 Elsevier SAS. All rights reserved. Keywords: Ca 2+ ; Cold-stress; Plant mitochondria; Potato tubers; –SH active reagents; SHAM 1. Introduction The complexity of the respiratory chain of plant mitochon- dria is partly due to the presence of an alternative pathway that, along with the conventional respiratory chain via cyto- chromes, catalyses electron reduction of oxygen to water. This alternative electron transport pathway branches at ubiquinone to a homodimer called alternative oxidase (AOX) that is inde- pendent of complexes III and IV, insensitive to inhibitors of these complexes, and sensitive to salicylhydroxamic acid (SHAM) [12,23]. This alternative pathway does not pump protons through the inner membrane and thus can be used to dissipate energy as heat, instead ofATP generation [18,21,24]. Indeed, the high expression of AOX in specific plant ther- mogenic tissues (e.g. Sauromatum guttatum) lead to the thought that this pathway was related only to heat production [24]. However, the presence of this pathway in other tissues has also been described [6,7,9,11,16,19,20,22], and AOX has been shown to be induced by stress conditions, such as patho- gen attack [7,20], senescence and fruit ripening [17], and chill- ing [2,5,23]. It has thus been suggested that the AOX path- way also operates to prevent the formation of reactive oxygen species in respiring mitochondria [9,12,17]. The expression of the AOX pathway in non-thermogenic plant tissues was firstly suggested to operate as an energy overflow mechanism when the cytochrome pathway is satu- Abbreviations: AOX, alternative oxidase; FCCP, carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone; HEPES, N-(2-hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid); NADH-DH, NADH dehydrogenase; PUMP, plant uncoupling mitochondrial protein; SHAM, salicylhydroxamic acid. * Corresponding author. Tel.: +55 41 361 1664; fax: +55 41 266 2042. E-mail address: egscarnieri@ufpr.br (E.G.S. Carnieri). Plant Physiology and Biochemistry 43 (2005) 61–67 www.elsevier.com/locate/plaphy 0981-9428/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.plaphy.2004.12.008