Effect of ischemia-reperfusion injury on the morphology of peroxisomes

We have previously demonstrated that ischemic injury changed the density of peroxisomes into two distinct peaks, one with a normal density (1.21 g/cm³; Peak I) and a second peak with a lighter density (1. 14 g/cm³; Peak II).We studied the peroxisomes from both peaks under the Electron microscope. Examination of peak I following ischemia showed loss of matrix proteins and damaged limiting membranes with leakage of DAB positive material in direct proportion to the duration of ischemia. Upon reperfusion of the ischemic liver Peak I showed more severe damage to the organelle. These observations clearly demonstrated that ischemia reperfusion injury causes structural damage to peroxisomes. Interestingly ultrastructural examination of Peak II following ischemia showed evidence of perisomal proliferation with budding of existing peroxisomes and the presence of micro peroxisomes (changes similar to those noted under conditions leading to perisomal proliferation). However, peak II following reperfusion showed only damaged organelle. These observations underline the importance of peroxisomes in the response of the cell to ischemia-reperfusion injury.     

Cadmium causes the oxidative modification of proteins in pea plants

In pea (Pisum sativum L.) leaves from plants grown in the presence of 50 µm CdCl₂ the oxidative production of carbonyl groups in proteins, the rate of protein degradation and the proteolytic activity were investigated. In leaf extracts the content of carbonyl groups measured by derivatization with 2,4‐dinitrophenylhydrazine (DNPH), was two‐fold higher in plants treated with Cd than in control plants. The identification of oxidized proteins was carried out by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of proteins derivatized with DNPH and immunochemical detection with an antibody against DNPH. The intensity of the reactive bands was higher in plants exposed to Cd than in controls. By using different antibodies some of the oxidized proteins were identified as Rubisco, glutathione reductase, manganese superoxide dismutase, and catalase. The incubation of leaf crude extracts with increasing H₂O₂ concentrations showed a progressive enhancement in carbonyl content and the pattern of oxidized proteins was similar to that found in Cd‐treated plants. Oxidized proteins were more efficiently degraded, and the proteolytic activity increased 20% due to the metal treatment. In peroxisomes purified from pea leaves a rise in the carbonyl content similar to that obtained in crude extracts from Cd‐treated plants was observed, but the functionality of the peroxisomal membrane was not apparently affected by Cd. Results obtained demonstrate the participation of both oxidative stress, probably mediated by H₂O₂, and proteolytic degradation in the mechanism of Cd toxicity in leaves of pea plants, and they appear to be involved in the Cd‐induced senescence previously reported in these plants.     

Determinants of the assembly,integrity and maintenance of the endoplasmic reticulum‐peroxisome tether

Organelle tethering and intercommunication are crucial for proper cell function. We previously described a tether between peroxisomes and the endoplasmic reticulum (ER) that acts in peroxisome population control in the yeast, Saccharomyces cerevisiae. Components of this tether are Pex3p, an integral membrane protein of both peroxisomes and the ER and Inp1p, a connector that links peroxisomes to the ER. Here, we report the analysis of random Inp1p mutants that enabled identification of regions in Inp1p required for the assembly and maintenance of the ER‐peroxisome tether. Interaction analysis between Inp1p mutants and known Inp1p‐binding proteins demonstrated that Pex3p and Inp1p do not constitute the sole components of the ER‐peroxisome tether. Deletion of these Inp1p interactors whose steady‐state localization is outside of ER‐peroxisome tethers affected peroxisome dynamics. Our findings are consistent with the presence of regulatory cues that act on ER‐peroxisome tethers and point to the existence of membrane contact sites between peroxisomes and organelles other than the ER.      

Peroxisomes sense and respond to environmental cues by regulating ROS and RNS signalling networks

Background Peroxisomes are highly dynamic, metabolically active organelles that used to be regarded as a sink for H₂O₂ generated in different organelles. However, peroxisomes are now considered to have a more complex function, containing different metabolic pathways, and they are an important source of reactive oxygen species (ROS), nitric oxide (NO) and reactive nitrogen species (RNS). Over-accumulation of ROS and RNS can give rise oxidative and nitrosative stress, but when produced at low concentrations they can act as signalling molecules.Scope This review focuses on the production of ROS and RNS in peroxisomes and their regulation by antioxidants. ROS production is associated with metabolic pathways such as photorespiration and fatty acid β-oxidation, and disturbances in any of these processes can be perceived by the cell as an alarm that triggers defence responses. Genetic and pharmacological studies have shown that photorespiratory H₂O₂ can affect nuclear gene expression, regulating the response to pathogen infection and light intensity. Proteomic studies have shown that peroxisomal proteins are targets for oxidative modification, S-nitrosylation and nitration and have highlighted the importance of these modifications in regulating peroxisomal metabolism and signalling networks. The morphology, size, number and speed of movement of peroxisomes can also change in response to oxidative stress, meaning that an ROS/redox receptor is required. Information available on the production and detection of NO/RNS in peroxisomes is more limited. Peroxisomal homeostasis is critical for maintaining the cellular redox balance and is regulated by ROS, peroxisomal proteases and autophagic processes.Conclusions Peroxisomes play a key role in many aspects of plant development and acclimation to stress conditions. These organelles can sense ROS/redox changes in the cell and thus trigger rapid and specific responses to environmental cues involving changes in peroxisomal dynamics as well as ROS- and NO-dependent signalling networks, although the mechanisms involved have not yet been established. Peroxisomes can therefore be regarded as a highly important decision-making platform in the cell, where ROS and RNS play a determining role.     

Physiology of pepper fruit and the metabolism of antioxidants: chloroplasts,mitochondria and peroxisomes

Background and Aims Pepper (Capsicum annuum) contains high levels of antioxidants, such as vitamins A and C and flavonoids. However, information on the role of these beneficial compounds in the physiology of pepper fruit remains scarce. Recent studies have shown that antioxidants in ripe pepper fruit play a key role in responses to temperature changes, and the redox state at the time of harvest affects the nutritional value for human consumption. In this paper, the role of antioxidant metabolism of pepper fruit during ripening and in the response to low temperature is addressed, paying particular attention to ascorbate, NADPH and the superoxide dismutase enzymatic system. The participation of chloroplasts, mitochondria and peroxisomes in the ripening process is also investigated.Scope and Results Important changes occur at a subcellular level during ripening of pepper fruit. Chloroplasts turn into chromoplasts, with drastic conversion of their metabolism, and the role of the ascorbate–glutathione cycle is essential. In mitochondria from red fruits, higher ascorbate peroxidase (APX) and Mn-SOD activities are involved in avoiding the accumulation of reactive oxygen species in these organelles during ripening. Peroxisomes, whose antioxidant capacity at fruit ripening is substantially affected, display an atypical metabolic pattern during this physiological stage. In spite of these differences observed in the antioxidative metabolism of mitochondria and peroxisomes, proteomic analysis of these organelles, carried out by 2-D electrophoresis and MALDI-TOF/TOF and provided here for the first time, reveals no changes between the antioxidant metabolism from immature (green) and ripe (red) fruits.Conclusions Taken together, the results show that investigation of molecular and enzymatic antioxidants from cell compartments, especially chloroplasts, mitochondria and peroxisomes, is a useful tool to study the physiology of pepper fruit, particularly in the context of expanding their shelf-life after harvest and in maintaining their nutritional value.     

The peroxisomal protein import machinery displays a preference for monomeric substrates

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.     

The role of peroxisomes in xylose alcoholic fermentation in the engineered Saccharomyces cerevisiae

Xylose is a second‐most abounded sugar after glucose in lignocellulosic hydrolysates and should be efficiently fermented for economically viable second‐generation ethanol production. Despite significant progress in metabolic and evolutionary engineering, xylose fermentation rate of recombinant Saccharomyces cerevisiae remains lower than that for glucose. Our recent study demonstrated that peroxisome‐deficient cells of yeast Ogataea polymorpha showed a decrease in ethanol production from xylose. In this work, we have studied the role of peroxisomes in xylose alcoholic fermentation in the engineered xylose‐utilizing strain of S. cerevisiae. It was shown that peroxisome‐less pex3Δ mutant possessed 1.5‐fold decrease of ethanol production from xylose. We hypothesized that peroxisomal catalase Cta1 may have importance for hydrogen peroxide, the important component of reactive oxygen species, detoxification during xylose alcoholic fermentation. It was clearly shown that CTA1 deletion impaired ethanol production from xylose. It was found that enhancing the peroxisome population by modulation the peroxisomal biogenesis by overexpression of PEX34 activates xylose alcoholic fermentation.     

Silencing of OsCV (chloroplast vesiculation) maintained photorespiration and N assimilation in rice plants grown under elevated CO2

High CO₂ concentrations stimulate net photosynthesis by increasing CO₂ substrate availability for Rubisco, simultaneously suppressing photorespiration. Previously, we reported that silencing the chloroplast vesiculation (cv) gene in rice increased source fitness, through the maintenance of chloroplast stability and the expression of photorespiration-associated genes. Because high atmospheric CO₂ conditions diminished photorespiration, we tested whether CV silencing might be a viable strategy to improve the effects of high CO₂ on grain yield and N assimilation in rice. Under elevated CO₂, OsCV expression was induced, and OsCV was targeted to peroxisomes where it facilitated the removal of OsPEX11-1 from the peroxisome and delivered it to the vacuole for degradation. This process correlated well with the reduction in the number of peroxisomes, the decreased catalase activity and the increased H₂O₂ content in wild-type plants under elevated CO₂. At elevated CO₂, CV-silenced rice plants maintained peroxisome proliferation and photorespiration and displayed higher N assimilation than wild-type plants. This was supported by higher activity of enzymes involved in NO₃⁻ and NH₄⁺ assimilation and higher total and seed protein contents. Co-immunoprecipitation of OsCV-interacting proteins suggested that, similar to its role in chloroplast protein turnover, OsCV acted as a scaffold, binding peroxisomal proteins.