It has recently been shown that cyanide-resistant respiration (CRR) is very common in Crabtree-negative yeasts (incapable of aerobic fermentation) and in non-fermentative yeasts. It is conferred by a salicylhydroxamic acid-sensitive alternative oxidase that transfers electrons from ubiquinol to oxygen, bypassing the cytochrome chain. An interesting finding is that, in general, whenever CRR is present, complex I is also present. In this article we briefly review the occurrence of CRR, the biochemistry and molecular biology of the alternative oxidase, and summarise the putative functions that have been attributed to this ubiquitous metabolic pathway, whose usefulness for the yeast cells still remains obscure.

Aims: To investigate the conditions that promote the expression of cyanide-resistant respiration (CRR) in the spoilage yeasts Pichia membranifaciens and Debaryomyces hansenii. Methods and Results: CRR was detected by sensitivity of oxygen consumption to salicylhydroxamic acid. It was absent in both yeasts in the early exponential phase, but was triggered by several stress situations. Starvation under aerobic conditions, decreasing pH or incubation of the culture in a narrow temperature range below the maximum temperature for growth promoted the emergence of CRR in both yeasts. In D. hansenii, CRR was also induced by 1AE5-2 mol l )1 NaCl. Although the presence of H 2 O 2 and menadione induced CRR, radical scavengers had no effect on the emergence of CRR. Also, the level of reactive oxygen species did not vary with the CRR activity.Conclusions: Under aerobic conditions, a respiratory pathway alternative to the cytochrome chain is triggered by stress conditions in P. membranifaciens and D. hansenii. Significance and Impact of the Study: The relationship between stress situations and CRR must be taken into account in studies on the performance of spoilage yeasts in the food processing environments where several forms of stress are common.

Cyanide-resistant respiration (CRR) is a widespread metabolic pathway among yeasts, that involves a mitochondrial alternative oxidase sensitive to salicylhydroxamic acid (SHAM). The physiological role of this pathway has been obscure. We used the yeasts Debaryomyces hansenii and Pichia membranifaciens to elucidate the involvement of CRR in energy conversion. In both yeasts the adenosine triphosphate (ATP) content was still high in the presence of antimycin A or SHAM, but decreased to low levels when both inhibitors were present simultaneously, indicating that CRR was involved in ATP formation. Also the mitochondrial membrane potential (Delta Psi(m)), monitored by fluorescent dyes, was relatively high in the presence of antimycin A and decreased upon addition of SHAM. In both yeasts the presence of complex I was confirmed by the inhibition of oxygen consumption in isolated mitochondria by rotenone. Comparing in the literature the occurrence of CRR and of complex I among yeasts, we found that CRR and complex I were simultaneously present in 12 out of 13 yeasts, whereas in six out of eight yeasts in which CRR was absent, complex I was also absent. Since three phosphorylating sites are active in the main respiratory chain and only one in CRR, we propose a role for this pathway in the fine adjustment of energy provision to the cell.

To investigate how the fatty acid composition of membrane lipids influences cell growth and mitochondrial respiration, in particular the expression and capacity of alternative oxidase (AOX), under cold stress, we used the Arabidopsis thaliana fad2 knockout and FAD3+ -overexpressing cultured cells lines affected in extrachloroplastic fatty acid desaturation activities. At 22 degrees C, fad2 mitochondria exhibited a low polyunsaturated fatty acid content and low protein to lipid ratio, while mitochondria from FAD3+ were enriched in linolenic acid and in total membrane protein. As a consequence, both mutants showed a higher membrane microviscosity than the wild type. After exposure to 9 degrees C, FAD3+ mitochondria exhibited lower microviscosity and lower rigidification upon a temperature downshift than fad2. Furthermore, the extent of reduction of cell growth and respiratiory rates in the phosphorylating state was positively related to the cold sensitivity of each cell line, being more pronounced in fad2 that in the wild type, whereas the stability of those parameters reflected the cold resistance of FAD3+. In contrast, an increase in AOX capacity was observed in the three cell lines at 9 degrees C. These inductions were correlated to AOX protein amounts and seem to result from an accumulation of AOX1c transcripts in the three cell lines and of AOX1a transcripts in wild-type and fad2 cells. The fact that there is no direct relationship between the degree of cold tolerance of each cell line and their ability to enhance their AOX capacity suggests that the participation of AOX in the response of Arabidopsis cells to cold stress does not necessarily favor cold tolerance.

Changes in mitochondrial respiration were investigated during cold storage of apple fruit (Malus domestica Borkh. cv. Reinette du Canada). Postharvest calcium treatment of the apples had no effect on the aspects of mitochondrial metabolism studied, suggesting that the role of calcium in fruit ripening is not related to respiration. A transient increase in mitochondrial oxidative capacity occurred during storage around day 80 after harvest. This climacteric burst was associated with an enhanced capacity of the alternative pathway, but not of the cytochrome pathway. The major factor determining this rise in alternative pathway capacity at the climacteric was the transient increase in the amount of alternative oxidase (AOX) protein. Antibodies raised against the Sauromatum guttatum enzyme recognized a reduced form product of ampproximately 37 kDa and a 67‐kDa band corresponding to the oxidized form of the protein. During cold storage of the apple fruit, alternative pathway capacity correlated with the levels of the AOX and was ampparently not dependent on major changes in the oxidation state of the enzyme. This is the first work to report that the respiratory climacteric and the induction of AOX are tightly and temporally linked.