João Daniel Arrabaça

Study of the effects of salicylic acid on soybean mitochondrial lipids and respiratory properties using the alternative oxidase as a stress-reporter protein.

Biotic and abiotic stresses can lead to modifications in the lipid composition of cell membranes. Although mitochondria appear to be implicated in stress responses, little is known about the membrane lipid changes that occur in these organelles in plants. Besides cytochrome c oxidase, plant mitochondria have an alternative oxidase (AOX) that accepts electrons directly from ubiquinol, dissipating energy as heat. AOX upregulation occurs under a variety of stresses and its induction by salicylic acid (SA) has been observed in different plant species. AOX was also suggested to be used as a functional marker for cell reprogramming under stress. In the present study, we have used etiolated soybean (Glycine max (L.) Merr. cv Cresir) seedlings to study the effects of SA treatment on the lipid composition and the respiratory properties of hypocotyl mitochondria. AOX expression was studied in detail, as a reporter protein, to evaluate whether modifications in mitochondrial energy metabolism were occurring. In mitochondria extracted from SA-treated seedlings, AOX capacity and protein contents increased. Both AOX1 and AOX2b transcripts accumulated in response to SA, but with different kinetics. A reduction in external NADH oxidation capacity was observed, whereas succinate respiration remained unchanged. The phospholipid composition of mitochondria remained similar in control and SA-treated plants, but a reduction in the relative amount of linolenic acid was observed in phosphatidylcholine, phosphatidylethanolamine and cardiolipin. The possible causes of the fatty acid modifications observed, and the implications for mitochondrial metabolism are discussed.

Involvement of AOX and UCP pathways in the post-harvest ripening of papaya fruits

Enhanced respiration during ripening in climacteric fruits is sometimes associated with an uncoupling between the ATP synthesis and the mitochondrial electron transport chain. While the participation of two energy-dissipating systems, one of which is mediated by the alternative oxidase (AOX) and the other mediated by the uncoupling protein (UCP), has been linked to fruit ripening, the relation between the activation of both mitochondrial uncoupling systems with the transient increase of ethylene synthesis (ethylene peak) remains unclear. To elucidate this question, ethylene emission and the two uncoupling (AOX and UCP) pathways were monitored in harvested papaya fruit during the ripening, from green to fully yellow skin. The results confirmed the typical climacteric behavior for papaya fruit: an initial increase in endogenous ethylene emission which reaches a maximum (peak) in the intermediate ripening stage, before finally declining to a basal level in ripe fruit. Respiration of intact fruit also increased and achieved higher levels at the end of ripening. On the other hand, in purified mitochondria extracted from fruit pulp the total respiration and respiratory control decrease while an increase in the participation of AOX and UCP pathways was markedly evident during papaya ripening. There was an increase in the AOX capacity during the transition from green fruit to the intermediate stage that accompanied the transient ethylene peak, while the O2 consumption triggered by UCP activation increased by 80% from the beginning to end stage of fruit ripening. Expression analyses of AOX (AOX1 and 2) and UCP (UCP1-5) genes revealed that the increases in the AOX and UCP capacities were linked to a higher expression of AOX1 and UCP (mainly UCP1) genes, respectively. In silico promoter analyses of both genes showed the presence of ethylene-responsive cis-elements in UCP1 and UCP2 genes. Overall, the data suggest a differential activation of AOX and UCP pathways in regulation related to the ethylene peak and induction of specific genes such as AOX1 and UCP1.

Fungistatic action of Aureobasidium pullulans on Penicillium expansum in “Rocha” pear: implications for oxidative stress during fruit storage

In “Rocha” pear, postharvest diseases, blue and grey moulds caused by Penicillium expansum and Botrytis cinerea, respectively, are particularly important. The control of postharvest decay has been based on the use of synthetic fungicides. However, there are risks to consumer health and the environment. In previous work, the antagonistic activity of the yeast-like fungus Aureobasidium pullulans (isolated from the surfaces of fruit, leaves and branches of “Rocha” pear trees), when applied as a wound treatment, was able to significantly reduce postharvest decay caused by P. expansum under storage conditions, and during simulated shelf-life. For more effective prevention and reduction of postharvest diseases in fruits it is necessary to understand the complex interactions in biological control between host, pathogen and antagonist, for which information is currently lacking. Thus, the aims of this study were to evaluate the biocontrol activity of the yeast-like fungus A. pullulans against P. expansum and to elucidate the resistance mechanism involved in the antagonistic activity of A. pullulans to oxidative stress. Fruits of Pyrus communis L. cv. Rocha were stored at −0.5°C, 95% relative humidity for 5.5 months. Fruit maturity was measured in terms of pericarp colour, sugar analysis, Streif index and flesh firmness. Assays of carbon dioxide and ethylene, electrolyte leakage, ascorbate, glutathione and antioxidant enzymes were used to evaluate oxidative stress. Furthermore, malondialdehyde, considered as a final product of lipid peroxidation, and the levels of hydrogen peroxide and methanesulfonic acid were determined and linked to oxidative stress impact on the lipid phase of cell membranes. During a 5-month period, fruit firmness, value of hue angle and colour, and Streif index changed significantly. Sucrose content decreased whereas fructose, glucose and sorbitol levels increased. After inoculation with A. pullulans and infection with P. expansum, a fungistatic, rather than fungicidal, effect was detected from 2.5 months onwards. Three months after storage, the higher superoxide dismutase activity and hydrogen peroxide levels caused by A. pullulans plus P. expansum were possibly associated with additional unsaturated fatty acids, as reflected by increased acyl lipid peroxidation. After five months of storage, in tissues inoculated with A. pullulans followed by infection with P. expansum, oxidative stress was linked to a general increase in the antioxidant peroxidase system activity. Glutathione and ascorbate content also increased along with the loss of cellular membrane selectivity. The results revealed a fungistatic effect of A pullulans on P. expansum. The prevalence of oxidative stress on “Rocha” pear wounds during the storage period was characterized, taking into account the interactions between A. pullulans and P. expansum. The information gained forms the basis for a biocontrol strategy, and the development of successful formulations for application in the orchard and after harvest. This strategy could eradicate and protect fruit against postharvest diseases, and is thus a promising alternative to the use of synthetic pesticides, being safe for the environment and for human health.

Genetically Modified Arabidopsis thaliana Cells Reveal the Involvement of the Mitochondrial Fatty Acid Composition in Membrane Basal and Uncoupling Protein-Mediated Proton Leaks

We investigated the role of membrane fatty acids in basal proton leaks and uncoupling protein (UCP)-dependent proton conductance in Arabidopsis mitochondria. Using wild-type cells, cold-sensitive fad2 mutant cells, deficient in omega-6-oleate desaturase, and cold-tolerant FAD3(+) transformant cells, overexpressing omega-3-linoleate desaturase, we showed that basal proton leak in the non-phosphorylating state was dependent on lipid composition. The extent of membrane proton leak was drastically reduced in the fad2 mutant, containing low amounts of polyunsaturated fatty acids. Conversely, this proton leak was higher in FAD3(+) mitochondria that exhibit a higher polyunsaturated fatty acid content and high protein to lipid ratio. The dependency of membrane leaks upon membrane potential was higher in FAD3(+) and lower in fad2. UCP content was higher in both the fad2 mutant and FAD3(+) transgenic lines compared with wild-type cells and so was the UCP activity, assayed by the reduction of phosphorylation yield (ADP/O) triggered by palmitate as UCP activator. This UCP assay was validated by measurements of UCP-proton leak in the non-phosphorylating state (flux-force relationships between proton flux and membrane potential). The potential uncoupling capacity of the UCP was high enough to allow the loss of respiratory control in the three genotypes. Taken together, the data reported here suggest that the cold tolerance of FAD3(+) cells and the cold sensitivity of fad2 cells are associated with changes in their mitochondrial membrane basal proton leaks, whereas differences in functional expression of UCP are not simply related to cold adaptation in Arabidopsis cells.

The Effects of Inhibitors of the B-C1 Complex on the Respiration of Mitochondria from Aged Potato Discs

The nature and function of the alternative, cyanide insensitive terminal oxidase, found in most plant tissues and in some microorganisms, has long been puzzling plant physiologists and biochemists.