A Role for RNS in the Communication of Plant Peroxisomes with Other Cell Organelles?

Plant peroxisomes are organelles with a very active participation in the cellular regulation of the metabolism of reactive oxygen species (ROS). However, during the last two decades peroxisomes have been shown to be also a relevant source of nitric oxide (NO) and other related molecules designated as reactive nitrogen species (RNS). ROS and RNS have been mainly associated to nitro-oxidative processes; however, some members of these two families of molecules such as H2O2, NO or S-nitrosoglutathione (GSNO) are also involved in the mechanism of signaling processes mainly through post-translational modifications. Peroxisomes interact metabolically with other cell compartments such as chloroplasts, mitochondria or oil bodies in different pathways including photorespiration, glyoxylate cycle or ß-oxidation, but peroxisomes are also involved in the biosynthesis of phytohormones including auxins and jasmonic acid (JA). This review will provide a comprehensive overview of peroxisomal RNS metabolism with special emphasis in the identified protein targets of RNS inside and outside these organelles. Moreover, the potential interconnectivity between peroxisomes and other plant organelles, such as mitochondria or chloroplasts, which could have a regulatory function will be explored, with special emphasis on photorespiration.

The Proteome of Fruit Peroxisomes: Sweet Pepper (Capsicum annuum L.) as a Model.

Despite of their economical and nutritional interest, the biology of fruits is still little studied in comparison with reports of other plant organs such as leaves and roots. Accordingly, research at subcellular and molecular levels is necessary not only to understand the physiology of fruits, but also to improve crop qualities. Efforts addressed to gain knowledge of the peroxisome proteome and how it interacts with the overall metabolism of fruits will provide tools to be used in breeding strategies of agricultural species with added value. In this work, special attention will be paid to peroxisomal proteins involved in the metabolism of reactive oxygen species (ROS) due to the relevant role of these compounds at fruit ripening. The proteome of peroxisomes purified from sweet pepper (Capsicum annuum L.) fruit is reported, where an iron-superoxide dismutase (Fe-SOD) was localized in these organelles, besides other antioxidant enzymes such as catalase and a Mn-SOD, as well as enzymes involved in the metabolism of carbohydrates, malate, lipids and fatty acids, amino acids, the glyoxylate cycle and in the potential organelles' movements.

Plant peroxisomes at the crossroad of NO and H2 O2 metabolism.

Plant peroxisomes are subcellular compartments involved in many biochemical pathways during the life cycle of a plant but also in the mechanism of response against adverse environmental conditions. These organelles have an active nitro-oxidative metabolism under physiological conditions but this could be exacerbated under stress situations. Furthermore, peroxisomes have the capacity to proliferate and also undergo biochemical adaptations depending on the surrounding cellular status. An important characteristic of peroxisomes is that they have a dynamic metabolism of reactive nitrogen and oxygen species (RNS and ROS) which generates two key molecules, nitric oxide (NO) and hydrogen peroxide (H2 O2 ). These molecules can exert signaling functions by means of post-translational modifications that affect the functionality of target molecules like proteins, peptides or fatty acids. This review provides an overview of the endogenous metabolism of ROS and RNS in peroxisomes with special emphasis on polyamine and uric acid metabolism as well as the possibility that these organelles could be a source of signal molecules involved in the functional interconnection with other subcellular compartments.

ROS Generation in Peroxisomes and its Role in Cell Signaling.

In plant cells, as in most eukaryotic organisms, peroxisomes are probably the major sites of intracellular H2O2 production, as a result of their essentially oxidative type of metabolism. In recent years, it has become increasingly clear that peroxisomes carry out essential functions in eukaryotic cells. The generation of the important messenger molecule hydrogen peroxide (H2O2) by animal and plant peroxisomes and the presence of catalase in these organelles has been known for many years, but the generation of superoxide radicals (O2·- ) and the occurrence of the metalloenzyme superoxide dismutase was reported for the first time in peroxisomes from plant origin. Further research showed the presence in plant peroxisomes of a complex battery of antioxidant systems apart from catalase. The evidence available of reactive oxygen species (ROS) production in peroxisomes is presented, and the different antioxidant systems characterized in these organelles and their possible functions are described. Peroxisomes appear to have a ROS-mediated role in abiotic stress situations induced by the heavy metal cadmium (Cd) and the xenobiotic 2,4-D, and also in the oxidative reactions of leaf senescence. The toxicity of Cd and 2,4-D has an effect on the ROS metabolism and speed of movement (dynamics) of peroxisomes. The regulation of ROS production in peroxisomes can take place by post-translational modifications of those proteins involved in their production and/or scavenging. In recent years, different studies have been carried out on the proteome of ROS metabolism in peroxisomes. Diverse evidence obtained indicates that peroxisomes are an important cellular source of different signaling molecules, including ROS, involved in distinct processes of high physiological importance, and might play an important role in the maintenance of cellular redox homeostasis.

ROS and RNS in plant physiology: an overview.

The production of reactive oxygen species (ROS) is the unavoidable consequence of aerobic life. ROS is a collective term that includes both oxygen radicals, like superoxide (O 2. -) and hydroxyl (·OH) radicals, and other non-radicals such as hydrogen peroxide (H2O2), singlet oxygen ((1)O2 or (1)Δg), etc. In plants, ROS are produced in different cell compartments and are oxidizing species, particularly hydroxyl radicals and singlet oxygen, that can produce serious damage in biological systems (oxidative stress). However, plant cells also have an array of antioxidants which, normally, can scavenge the excess oxidants produced and so avoid deleterious effects on the plant cell bio-molecules. The concept of 'oxidative stress' was re-evaluated in recent years and the term 'oxidative signalling' was created. This means that ROS production, apart from being a potentially harmful process, is also an important component of the signalling network that plants use for their development and for responding to environmental challenges. It is known that ROS play an important role regulating numerous biological processes such as growth, development, response to biotic and environmental stresses, and programmed cell death. The term reactive nitrogen species (RNS) includes radicals like nitric oxide (NO· ) and nitric dioxide (NO2.), as well as non-radicals such as nitrous acid (HNO2) and dinitrogen tetroxide (N2O4), among others. RNS are also produced in plants although the generating systems have still not been fully characterized. Nitric oxide (NO·) has an important function as a key signalling molecule in plant growth, development, and senescence, and RNS, like ROS, also play an important role as signalling molecules in the response to environmental (abiotic) stress. Similarly, NO· is a key mediator, in co-operation with ROS, in the defence response to pathogen attacks in plants. ROS and RNS have been demonstrated to have an increasingly important role in biology and medicine.

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.

Spatial and temporal regulation of the metabolism of reactive oxygen and nitrogen species during the early development of pepper (Capsicum annuum) seedlings.

BACKGROUND AND AIMS: The development of seedlings involves many morphological, physiological and biochemical processes, which are controlled by many factors. Some reactive oxygen and nitrogen species (ROS and RNS, respectively) are implicated as signal molecules in physiological and phytopathological processes. Pepper (Capsicum annuum) is a very important crop and the goal of this work was to provide a framework of the behaviour of the key elements in the metabolism of ROS and RNS in the main organs of pepper during its development. METHODS: The main seedling organs (roots, hypocotyls and green cotyledons) of pepper seedlings were analysed 7, 10 and 14 d after germination. Activity and gene expression of the main enzymatic antioxidants (catalase, ascorbate-glutathione cycle enzymes), NADP-generating dehydrogenases and S-nitrosoglutathione reductase were determined. Cellular distribution of nitric oxide ((·)NO), superoxide radical (O2 (·-)) and peroxynitrite (ONOO(-)) was investigated using confocal laser scanning microscopy. KEY RESULTS: The metabolism of ROS and RNS during pepper seedling development was highly regulated and showed significant plasticity, which was co-ordinated among the main seedling organs, resulting in correct development. Catalase showed higher activity in the aerial parts of the seedling (hypocotyls and green cotyledons) whereas roots of 7-d-old seedlings contained higher activity of the enzymatic components of the ascorbate glutathione cycle, NADP-isocitrate dehydrogenase and NADP-malic enzyme. CONCLUSIONS: There is differential regulation of the metabolism of ROS, nitric oxide and NADP dehydrogenases in the different plant organs during seedling development in pepper in the absence of stress. The metabolism of ROS and RNS seems to contribute significantly to plant development since their components are involved directly or indirectly in many metabolic pathways. Thus, specific molecules such as H2O2 and NO have implications for signalling, and their temporal and spatial regulation contributes to the success of seedling establishment.

Role of peroxisomes as a source of reactive oxygen species (ROS) signaling molecules.

Peroxisomes are very dynamic and metabolically active organelles and are a very important source of reactive oxygen species (ROS), H2O2, O2 (.-) and · OH, which are mainly produced in different metabolic pathways, including fatty acid ß-oxidation, photorespiration, nucleic acid and polyamine catabolism, ureide metabolism, etc. ROS were originally associated to oxygen toxicity; however, these reactive species also play a central role in the signaling network regulating essential processes in the cell. Peroxisomes have the capacity to rapidly produce and scavenge H2O2 and O2 (.-) which allows to regulate dynamic changes in ROS levels. This fact and the plasticity of these organelles, which allows adjusting their metabolism depending on different developmental and environmental cues, makes these organelles play a central role in cellular signal transduction. The use of catalase and glycolate oxidase loss-of-function mutants has allowed to study the consequences of changes in the levels of endogenous H2O2 in peroxisomes and has improved our knowledge of the transcriptomic profile of genes regulated by peroxisomal ROS. It is now known that peroxisomal ROS participate in more complex signaling networks involving calcium, hormones, and redox homeostasis which finally determine the response of plants to their environment.

Peroxisomes as cell generators of reactive nitrogen species (RNS) signal molecules.

Nitric oxide is a gaseous free radical with a wide range of direct and indirect actions in plant cells. However, the enzymatic sources of NO and its subcellular localization in plants are still under debate. Among the different subcellular compartments where NO has been found to be produced, peroxisomes are the best characterized since in these organelles it has been demonstrated the presence of NO and it has been biochemically characterized a L-arginine-dependent nitric oxide synthase activity. This chapter summarizes the present knowledge of the NO metabolism and its derived reactive nitrogen species (RNS) in plant peroxisomes and how this gaseous free radical is involved in natural senescence, and is released to the cytosol under salinity stress conditions acting as a signal molecule.

Advances in Melanoma: From Genetic Insights to Therapeutic Innovations.

Advances in melanoma research have unveiled critical insights into its genetic and molecular landscape, leading to significant therapeutic innovations. This review explores the intricate interplay between genetic alterations, such as mutations in BRAF, NRAS, and KIT, and melanoma pathogenesis. The MAPK and PI3K/Akt/mTOR signaling pathways are highlighted for their roles in tumor growth and resistance mechanisms. Additionally, this review delves into the impact of epigenetic modifications, including DNA methylation and histone changes, on melanoma progression. The tumor microenvironment, characterized by immune cells, stromal cells, and soluble factors, plays a pivotal role in modulating tumor behavior and treatment responses. Emerging technologies like single-cell sequencing, CRISPR-Cas9, and AI-driven diagnostics are transforming melanoma research, offering precise and personalized approaches to treatment. Immunotherapy, particularly immune checkpoint inhibitors and personalized mRNA vaccines, has revolutionized melanoma therapy by enhancing the body's immune response. Despite these advances, resistance mechanisms remain a challenge, underscoring the need for combined therapies and ongoing research to achieve durable therapeutic responses. This comprehensive overview aims to highlight the current state of melanoma research and the transformative impacts of these advancements on clinical practice.

Antioxidant systems from Pepper (Capsicum annuum L.): involvement in the response to temperature changes in ripe fruits.

Sweet pepper is susceptible to changes in the environmental conditions, especially temperatures below 15 °C. In this work, two sets of pepper fruits (Capsicum annuum L.) which underwent distinct temperature profiles in planta were investigated. Accordingly, two harvesting times corresponding to each set were established: Harvest 1, whose fruits developed and ripened at 14.9 °C as average temperature; and Harvest 2, with average temperature of 12.4 °C. The oxidative metabolism was analyzed in all fruits. Although total ascorbate content did not vary between Harvests, a shift from the reduced to the oxidized form (dehydroascorbate), accompanied by a higher ascorbate peroxidase activity, was observed in Harvest 2 with respect to Harvest 1. Moreover, a decrease of the ascorbate-generating enzymatic system, the γ-galactono-lactone dehydrogenase, was found at Harvest 2. The activity values of the NADP-dependent dehydrogenases analyzed seem to indicate that a lower NADPH synthesis may occur in fruits which underwent lower temperature conditions. In spite of the important changes observed in the oxidative metabolism in fruits subjected to lower temperature, no oxidative stress appears to occur, as indicated by the lipid peroxidation and protein oxidation profiles. Thus, the antioxidative systems of pepper fruits seem to be involved in the response against temperature changes.

Metabolism of reactive oxygen species and reactive nitrogen species in pepper (Capsicum annuum L.) plants under low temperature stress.

Low temperature is an environmental stress that affects crop production and quality and regulates the expression of many genes, and the level of a number of proteins and metabolites. Using leaves from pepper (Capsicum annum L.) plants exposed to low temperature (8 °C) for different time periods (1 to 3 d), several key components of the metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were analysed. After 24 h of exposure at 8 °C, pepper plants exhibited visible symptoms characterized by flaccidity of stems and leaves. This was accompanied by significant changes in the metabolism of RNS and ROS with an increase of both protein tyrosine nitration (NO(2) -Tyr) and lipid peroxidation, indicating that low temperature induces nitrosative and oxidative stress. During the second and third days at low temperature, pepper plants underwent cold acclimation by adjusting their antioxidant metabolism and reverting the observed nitrosative and oxidative stress. In this process, the levels of the soluble non-enzymatic antioxidants ascorbate and glutathione, and the activity of the main NADPH-generating dehydrogenases were significantly induced. This suggests that ascorbate, glutathione and the NADPH-generating dehydrogenases have a role in the process of cold acclimation through their effect on the redox state of the cell.

Differential response of young and adult leaves to herbicide 2,4-dichlorophenoxyacetic acid in pea plants: role of reactive oxygen species.

In this work the differential response of adult and young leaves from pea (Pisum sativum L.) plants to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) (23 mm) applied by foliar spraying was investigated. The concentration of 2,4-D (23 mm) and the time of treatment (72 h) were previously optimized in order to visualize its toxic effects on pea plants. Under these conditions, the herbicide induced severe disturbances in mesophyll cells structure and proliferation of vascular tissue in young leaves and increased acyl-CoA oxidase (ACX), xanthine oxidase (XOD) and lipoxygenase (LOX) activities in young leaves, and only ACX and LOX in adult leaves. This situation produced reactive oxygen species (ROS) over-accumulation favoured by the absence of significant changes in the enzymatic antioxidants, giving rise to oxidative damages to proteins and membrane lipids. An increase of ethylene took place in both young and adult leaves and the induction of genes encoding the stress proteins, PRP4A and HSP 71,2, was observed mainly in young leaves. These results suggest that ROS overproduction is a key factor in the effect of high concentrations of 2,4-D, and ROS can trigger a differential response in young and adult leaves, either epinasty development in young leaves or senescence processes in adult tissues.

Peroxisomes as a cellular source of reactive nitrogen species signal molecules.

Peroxisomes are single membrane-bounded subcellular organelles with an essentially oxidative type of metabolism and are probably the major sites of intracellular H(2)O(2) production. These organelles also generate superoxide radicals (O(2)(-)) and besides catalase they have a complex battery of antioxidative enzymes. In recent years the existence of l-arginine-dependent nitric oxide synthase (NOS) activity and the generation of the reactive nitrogen species (RNS) nitric oxide (NO) have been demonstrated in plant peroxisomes. The inter-cellular and intracellular NO carrier S-nitrosoglutathione (GSNO) can be generated inside peroxisomes and the presence of this RNS has been demonstrated in peroxisomes from several plant species. This review analyzes the available evidence concerning the properties of the NOS activity and the generation of the RNS messengers NO and GSNO in peroxisomes in the context of the cellular function of these organelles as a source of RNS signaling molecules. The important physiological functions displayed by NO and other RNS in intra- and inter-cellular communication in different organisms indicate that more attention should be payed to the RNS signaling function of peroxisomes in human, animal and fungal cells, where it is very likely that similar mechanisms to those found in plant peroxisomes are also operative.

Proteome of plant peroxisomes: new perspectives on the role of these organelles in cell biology.

Peroxisomes are cell organelles bounded by a single membrane with a basically oxidative metabolism. Peroxisomes house catalase and H(2)O(2)-producing flavin-oxidases as the main protein constituents. However, since their discovery in early fifties, a number of new enzymes and metabolic pathways have been reported to be also confined to these organelles. Thus, the presence of exo- and endo-peptidases, superoxide dismutases, the enzymes of the plant ascorbate-glutathione cycle plus ascorbate and glutathione, several NADP-dehydrogenases, and also L-arginine-dependent nitric oxide synthase activity has evidenced the relevant role of these organelles in cell physiology. In recent years, the study of new functions of peroxisomes has become a field of intensive research in cell biology, and these organelles have been proposed to be a source of important signal molecules for different transduction pathways. In plants, peroxisomes participate in seed germination, leaf senescence, fruit maturation, response to abiotic and biotic stress, photomorphogenesis, biosynthesis of the plant hormones jasmonic acid and auxin, and in cell signaling by reactive oxygen and nitrogen species (ROS and RNS, respectively). In order to decipher the nature and specific role of the peroxisomal proteins in these processes, several approaches including in vivo and in vitro import assays and generation of mutants have been used. In the last decade, the development of genomics and the report of the first plant genomes provided plant biologists a powerful tool to assign to peroxisomes those proteins which harbored any of the two peroxisomal targeting signals (PTS, either PTS1 or PTS2) described so far. Unfortunately, those molecular approaches could not give any response to those proteins previously localized in plant peroxisomes by classical biochemical and cell biology methods that did not contain any PTS. However, more recently, proteomic studies of highly purified organelles have provided evidence of the presence in peroxisomes of new proteins not previously reported. Thus, the contribution of proteomic approaches to the biology of peroxisomes is essential, not only for elucidation of the mechanisms involved in the import of the PTS1- and PTS2-independent proteins, but also to the understanding of the role of these organelles in the cell physiology of plant growth and development.

NADP-dehydrogenases from pepper fruits: effect of maturation.

NADPH is an important molecule in the redox balance of the cell. Pepper fruits are the second worldwide consumable vegetables and exhibit different phenotypes after maturation. In this paper, two pepper cultivars were studied: Vergasa whose fruits shift from green to red after maturation, and Biela that shifts to yellow. Using fresh fruits from the same plants of the two cultivars at distinct maturation stages, the activity and gene expression of the main NADPH-generating dehydrogenases was studied. The activity analysis of the main NADP-dehydrogenases, glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), NADP-isocitrate dehydrogenase (NADP-ICDH) and NADP-malic enzyme (NADP-ME), showed that, except for the G6PDH, all the activities were enhanced (54-100%) in the mature pepper fruits from both cultivars (red or yellow) with respect to green pepper fruits. The content of NADPH and NADP in the mature fruits of both cultivars showed a noteworthy increase with respect to green fruits. For the transcript analysis, a partial cDNA of each NADP-dehydrogenase was obtained, and the NADP-ME was the only NADP-dehydrogenase that showed a significant induction. The increase in the content of NADPH in mature fruits because of the enhanced activity of NADP-dehydrogenases suggests that these NADPH-generating enzymes could be involved in the maturation of pepper fruits.

Metabolism of reactive nitrogen species in pea plants under abiotic stress conditions.

Nitric oxide (*NO) is a key signaling molecule in different physiological processes of animals and plants. However, little is known about the metabolism of endogenous *NO and other reactive nitrogen species (RNS) in plants under abiotic stress conditions. Using pea plants exposed to six different abiotic stress conditions (high light intensity, low and high temperature, continuous light, continuous dark and mechanical wounding), several key components of the metabolism of RNS including the content of *NO, S-nitrosothiols (RSNOs) and nitrite plus nitrate, the enzyme activities of l-arginine-dependent nitric oxide synthase (NOS) and S-nitrosogluthathione reductase (GSNOR), and the profile of protein tyrosine nitration (NO(2)-Tyr) were analyzed in leaves. Low temperature was the stress that produced the highest increase of NOS and GSNOR activities, and this was accompanied by an increase in the content of total *NO and S-nitrosothiols, and an intensification of the immunoreactivity with an antibody against NO(2)-Tyr. Mechanical wounding, high temperature and light also had a clear activating effect on the different indicators of RNS metabolism in pea plants. However, the total content of nitrite and nitrate in leaves was not affected by any of these stresses. Considering that protein tyrosine nitration is a potential marker of nitrosative stress, the results obtained suggest that low and high temperature, continuous light and high light intensity are abiotic stress conditions that can induce nitrosative stress in pea plants.

Localization of S-nitrosothiols and assay of nitric oxide synthase and S-nitrosoglutathione reductase activity in plants.

The study of the metabolism of nitric oxide and other reactive nitrogen species (RNS) in plants has been the subject of intensive work in the last decade due to the relevance of these molecules in the physiology and biochemistry of plants. However, there are still many methodological limitations in the specific and accurate determination and localization of RNS. This chapter describes several biochemical and cellular methods demonstrated to be useful for this purpose in different plant tissues. These methods are the determination of L-arginine-dependent nitric oxide synthase and S-nitrosoglutathione reductase activities, as well as cellular localization by confocal laser-scanning microscopy of S-nitrosothiols, particularly S-nitrosoglutathione. These approaches can help advance the knowledge of the function of RNS in plant cells.

Imaging of reactive oxygen species and nitric oxide in vivo in plant tissues.

During the last decades there has been a growing interest in the study of reactive oxygen species (ROS) and nitric oxide (NO) production in plant tissues and their role in signaling and cellular response to biotic and abiotic stress conditions. Despite growing molecular data on this subject, less attention has been paid to the topological distribution of ROS and NO production in plant tissues. Knowledge of the contribution of different cells to the accumulation of ROS and NO is important to get deeper insights into the cellular response of plants to adverse conditions. This chapter focuses on the imaging of ROS and NO accumulation in vivo in plant tissues by confocal laser microscopy using specific fluorescent probes.