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The aim of this work was to evaluate the bioethanol productivity in an unconventional bioreactor assisted by extremely low frequency (ELF) - electromagnetic field and elucidate the biophysical mechanism of action by which ELF magnetic fields improve the bioethanol production by S. cerevisiae. Fermentations were carried out under axial field lines at 10 mT magnetic flux density (B), using three different recycling arrangements (spiral-shape tube, u-shape tube and whole bioreactor) in a closed loop. Fermentation kinetics were monitored by cell growth, substrate consumption, ethanol and by-product formation. In addition, electrophysiological measurements of the H+ ion fluxes were carried out in yeast cells sampled at different fermentation stages. ELF magnetic fields increased the glucose uptake, bioethanol production and H+ efflux, shortening in 2 h the fermentation time. The greatest effects of the ELF magnetic fields were obtained in the whole bioreactor arrangement, reaching an average increase of 33% in the bioethanol production. The results are consistent with a stimulatory effect of ELF magnetic fields on the plasma membrane H+-ATPase activity, as indicated by the specific increase of the vanadate-sensitive component of the yeast cells H+ efflux, providing a new biophysical mechanism of action for the biological effect of magnetic fields.

Mitochondria are the major organelles of energy production; however, active mitochondria can decline their energetic role and show a dysfunctional status. Mitochondrial dysfunction was induced by high non-physiological level of L-galactone-1,4-lactone (L-GalL), the precursor of ascorbate (AsA), in plant mitochondria. The dysfunction induced by L-GalL was associated with the fault in the mitochondrial electron partition and reactive oxygen species (ROS) over-production. Using mitochondria from RNAi-plant lines harbouring silenced L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activity, it was demonstrated that such dysfunction is dependent on this enzyme activity. The capacity of alternative respiration was strongly decreased by L-GalL, probably mediated by redox-inactivation of the alternative oxidase (AOX) enzyme. Although, alternative respiration was shown to be the key factor that helps support AsA synthesis in dysfunctional mitochondria. Experiments with respiratory inhibitors showed that ROS formation and mitochondrial dysfunction were more associated with the decline in the activities of COX (cytochrome oxidase) and particularly AOX than with the lower activities of respiratory complexes I and III. The application of high L-GalL concentrations induced proteomic changes that indicated alterations in proteins related to oxidative stress and energetic status. However, supra-optimal L-GalL concentration was not deleterious for plants. Instead, the L-GalLDH activity could be positive. Indeed, it was found that wild type plants performed better growth than L-GalLDH-RNAi plants in response to high non-physiological L-GalL concentrations.

Iron is an essential nutrient but is toxic in excess mainly under acidic conditions. Yeasts have emerged as low cost, highly efficient soil inoculants for the decontamination of metal-polluted areas, harnessing an increasing understanding of their metal tolerance mechanisms. Here, we investigated the effects of extracellular iron and acid pH stress on the dimorphism of Yarrowia lipolytica. Its growth was unaffected by 1 or 2 mM FeSO4, while a strong cellular iron accumulation was detected. However, the iron treatments decreased the hyphal length and number, mainly at 2 mM FeSO4 and pH 4.5. Inward cell membrane H+ fluxes were found at pH 4.5 and 6.0 correlated with a pH increase at the cell surface and a conspicuous yeast-to-hypha transition activity. Conversely, a remarkable H+ efflux was detected at pH 3.0, related to the extracellular microenvironment acidification and inhibition of yeast-to-hypha transition. Iron treatments intensified H+ influxes at pH 4.5 and 6.0 and inhibited H+ efflux at pH 3.0. Moreover, iron treatments inhibited the expression and activities of the plasma membrane H+-ATPase, with the H+ transport inhibited to a greater extent than the ATP hydrolysis, suggesting an iron-induced uncoupling of the pump. Our data indicate that Y. lipolytica adaptations to high iron and acidic environments occur at the expense of remodelling the yeast morphogenesis through a cellular pH modulation by H+-ATPases and H+ coupled transporters, highlighting the capacity of this non-conventional yeast to accumulate high amounts of iron and its potential application for bioremediation.

Attempts to improve the ascorbate (AsA) content of plants are still dealing with the limited understanding of why exists a wide variability of this powerful anti-oxidant molecule in different plant sources, species and environmental situations. In plant mitochondria, the last step of AsA synthesis is catalyzed by the enzyme L-galactone-1,4-lactone dehydrogenase (L-GalLDH). By using GalLDH-RNAi silencing plant lines, biochemical and proteomic approaches, we here discovered that, in addition to accumulate this antioxidant, mitochondria synthesize AsA to down-regulate the respiratory activity and the cellular energy provision. The work reveals that the AsA synthesis pathway within mitochondria is a branched electron transfer process that channels electrons towards the alternative oxidase, interfering with conventional electron transport. It was unexpectedly found that significant hydrogen peroxide is generated during AsA synthesis, which affects the AsA level. The induced AsA synthesis shows proteomic alterations of mitochondrial and extra-mitochondrial proteins related to oxidative and energetic metabolism. The most identified proteins were known components of plant responses to high light acclimation, programmed cell death, oxidative stress, senescence, cell expansion, iron and phosphorus starvation, different abiotic stress/pathogen attack responses and others. We propose that changing the electron flux associated with AsA synthesis might be part of a new mechanism by which the L-GalLDH enzyme would adapt plant mitochondria to fluctuating energy demands and redox status occurring under different physiological contexts., Instituto de Fisiología Vegetal

At relatively low concentrations, the element manganese (Mn) is essential for plant metabolism, especially for photosynthesis and as an enzyme antioxidant cofactor. However, industrial and agricultural activities have greatly increased Mn concentrations, and thereby contamination, in soils. We tested whether and how growth of Pisolithus tinctorius is influenced by Mn and glucose and compare the activities of oxidative stress enzymes as biochemical markers of Mn stress. We also compared nutrient accumulation, ecophysiology, and biochemical responses in Eucalyptus grandis which had been colonized by the ectomycorrhizal Pisolithus tinctorius with those which had not, when both were exposed to increasing Mn concentrations. In vitro experiments comprised six concentrations of Mn in three concentrations of glucose. In vivo experiments used plants colonized by Pisolithus tinctorius, or not colonized, grown with three concentrations of Mn (0, 200, and 1000 μM). We found that fungal growth and glucose concentration were correlated, but these were not influenced by Mn levels in the medium. The anti-oxidative enzymes catalase and glutathione S-transferase were both activated when the fungus was exposed to Mn. Also, mycorrhizal plants grew more and faster than non-mycorrhizal plants, whatever Mn exposure. Photosynthesis rate, intrinsic water use efficiency, and carboxylation efficiency were all inversely correlated with Mn concentration. Thus, we originally show that the ectomycorrhizal fungus provides protection for its host plants against varying and potentially toxic concentrations of Mn.

Several pollutants interfere with plant metabolism and heavy metals (HMs) are one of the most common. The HM toxicity can cause plant growth retardation, inhibition of photosynthesis, induction and inhibition of enzymes, DNA damage, and the generation of oxidative stress. To avoid toxicity from HMs, plants developed a range of different response mechanisms acting in additive and/or synergistic ways. The plant protection against metal toxicity occurs through control of root uptake of metal and metal transport within the plant. Proteins such as ferritin, metallothioneins, and glutathione-derived peptides (phytochelatins) and organic molecules such as organic acids and phytate play an important role in plant storage and detoxification process in metal homeostasis; when these systems are overloaded, oxidative stress defense mechanisms are activated. Understanding the functioning of these mechanisms in climate change is of utmost importance to understanding the process of adaptation of plants to environmental changes and their use in bioremediation process. This chapter chapter briefly summarizes some recent aspects of biological, molecular, and proteomic mechanisms concerning the responses of plants to HMs.

Despite the substrate specificity has not yet been clarified for the putative cation pumps belonging to the P5A-type ATPase subfamily, compelling evidences have increasingly been accumulated about its crucial physiological role in Ca2+ homeostasis and signal transduction. The aim of this review is to discuss some of the most relevant functional data available, which establish a clear relationship among different phenomena underlying or depending on tightly modulations of cellular Ca2+ changes and the functional expression of P5A-ATPases. Issues related to the different substrate propositions and structural analysis will only be mentioned, since they have thoroughly been explored in recent reviews. The relevance of critical biochemical characterizations in the completion of the most frequent molecular approaches in the elucidation of the key physiological role of the P5A-type ATPases will be highlighted, whose evolution seems to be tightly integrated to the origin of eukaryotes.