No abstract

The highly halotolerant yeast Debaryomyces hansenii when grown in the presence of 2M NaCl, increased the expression of ARO4 which is involved in the biosynthesis of aromatic amino acids. The function of the isolated gene was verified by complementation of a Saccharomyces cerevisiae null mutant, aro4 , restoring the specific activity of the enzyme (a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase) to wild-type levels. DhARO4 transcript expression under high salinity was stimulated at the beginning of the exponential growth phase. As the DhARO4 promoter region presents putative GCRE and CRE sequences, its expression was evaluated under conditions of NaCl stress and amino acid starvation, showing similar expression levels for either condition. The combined effect of both stressors resulted in a further increase in transcript levels over the singly added stressors, indicating independent stimulatory events. Our results support the hypothesis that high salinity and amino acid availability are physiologically interconnected.

It has been previously reported that growth of Debaryomyces hansenii in 2 M NaCl induced the expression of ARO4. This gene codifies for DhAro4p, involved in the synthesis of the amino acid tyrosine. In this work we studied the activity of DhAro4p upon salt stress; a higher activity was observed in cells grown with 2 M NaCl, but tyrosine levels were not increased. On the other hand, the addition of tyrosine to the saline medium significantly enhanced the growth of D. hansenii. It was found that the oxidized form of tyrosine, 3-nitrotyrosine, increased in the presence of salt. Since NaCl protects against oxidative stress in D. hansenii (Navarrete et al., 2009), we propose that a protective pathway is the de novo synthesis of tyrosine and its immediate oxidation to 3-nitrotyrosine to counteract oxidative stress generated by salt stress, so we measured the production of reactive oxygen species (ROS) and nitric oxide (NO À ) in D. hansenii after growing in 2 M NaCl. Results showed the presence of NO À and the increased production of ROS; this is probably due to an increased respiratory activity in the cells grown in the presence of salt. Our results demonstrate that upon salt stress D hansenii responds to oxidative stress via the transcriptional activation of specific genes such as DhARO4.

Increased membrane unsaturation has been associated with shorter longevity due to higher sensitivity to lipid peroxidation (LP) leading to enhanced mitochondrial dysfunction and ROS overproduction. However, the role of LP during aging has been put in doubt along with the participation of electron leak at the electron transport chain (ETC) in ROS generation in aged organisms. Thus, to test these hypothesis and gain further information about how minimizing LP preserves ETC function during aging, we studied the effects of α-linolenic acid (C18:3) on in situ mitochondrial ETC function, ROS production and viability of chronologically aged cells of S. cerevisiae, whose membranes are intrinsically resistant to LP due to the lack of PUFA. Increased sensitivity to LP was observed in cells cultured with C18:3 at 6 days of aging. This was associated with higher viability loss, dissipated membrane potential, impaired respiration and increased ROS generation, being these effects more evident at 28 days. However, at this point, lower sensitivity to LP was observed without changes in the membrane content of C18:3, suggesting the activation of a mechanism counteracting LP. The cells without C18:3 display better viability and mitochondrial functionality with lower ROS generation even at 28 days of aging and this was attributed to full preservation of complex III activity. These results indicate that the presence of PUFA in membranes enhances ETC dysfunction and electron leak and suggest that complex III is crucial to preserve membrane potential and to maintain a low rate of ROS production during aging.