Luis A. del Río

Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells

The important role of plant peroxisomes in a variety of metabolic reactions such as photorespiration, fatty acid beta-oxidation, the glyoxylate cycle and generation-degradation of hydrogen peroxide is well known. In recent years, the presence of a novel group of enzymes, mainly involved in the metabolism of oxygen free-radicals, has been shown in peroxisomes. In addition to hydrogen peroxide, peroxisomes can generate superoxide-radicals and nitric oxide, which are known cellular messengers with a variety of physiological roles in intra- and inter-cellular communication. Nitric oxide and hydrogen peroxide can permeate the peroxisomal membrane and superoxide radicals can be produced on the cytosolic side of the membrane. The signal molecule-generating capacity of peroxisomes can have important implications for cellular metabolism in plants, particularly under biotic and abiotic stress.

Cellular Response of Pea Plants to Cadmium Toxicity: Cross Talk between Reactive Oxygen Species, Nitric Oxide, and Calcium

Cadmium (Cd) toxicity has been widely studied in different plant species; however, the mechanism involved in its toxicity as well as the cell response against the metal have not been well established. In this work, using pea (Pisum sativum) plants, we studied the effect of Cd on antioxidants, reactive oxygen species (ROS), and nitric oxide (NO) metabolism of leaves using different cellular, molecular, and biochemical approaches. The growth of pea plants with 50 μm CdCl2 affected differentially the expression of superoxide dismutase (SOD) isozymes at both transcriptional and posttranscriptional levels, giving rise to a SOD activity reduction. The copper/zinc-SOD down-regulation was apparently due to the calcium (Ca) deficiency induced by the heavy metal. In these circumstances, the overproduction of the ROS hydrogen peroxide and superoxide could be observed in vivo by confocal laser microscopy, mainly associated with vascular tissue, epidermis, and mesophyll cells, and the production of superoxide radicals was prevented by exogenous Ca. On the other hand, the NO synthase-dependent NO production was strongly depressed by Cd, and treatment with Ca prevented this effect. Under these conditions, the pathogen-related proteins PrP4A and chitinase and the heat shock protein 71.2, were up-regulated, probably to protect cells against damages induced by Cd. The regulation of these proteins could be mediated by jasmonic acid and ethylene, whose contents increased by Cd treatment. A model is proposed for the cellular response to long-term Cd exposure consisting of cross talk between Ca, ROS, and NO.

Reactive oxygen species and reactive nitrogen species in peroxisomes. Production, scavenging, and role in cell signaling

Peroxisomes can be broadly defined as subcellular organelles bounded by a single membrane that contain as basic enzymatic constituents catalase and hydrogen peroxide (H2O2)-producing flavin oxidases and occur in almost all eukaryotic cells ([Baker and Graham, 2002][1]). In recent years, it has

Localization of Nitric-oxide Synthase in Plant Peroxisomes

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca2+-dependent NOS activity of 5.61 nmol ofl-[3H]citrulline mg−1 protein min−1 was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60–90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.

Plant proteases, protein degradation, and oxidative stress: role of peroxisomes

Growth and development in all organisms occur as a result of an overall balance between synthesis and proteolysis. In plants, protein degradation is a crucial mechanism in some developmental stages such as germination, morphogenesis and cell biogenesis, senescence, and programmed cell death. In this work, the main proteases that take part in these processes are reviewed. Proteolysis is also an important component together with protein oxidation in oxidative stress situations induced by senescence and heavy metals. The presence of exo- and endoproteolytic activity in plant peroxisomes is analyzed, and the role of peroxisomal proteases in different physiological events that take place under oxidative stress situations is discussed.

Cellular and Subcellular Localization of Endogenous Nitric Oxide in Young and Senescent Pea Plants

The cellular and subcellular localization of endogenous nitric oxide (NO˙) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO˙ was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO˙ generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)2 and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO˙ was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO˙, with g = 2.05 and aN = 12.8 G, was detected in peroxisomes. By fluorometry, NO˙ was also found in these organelles, and the level measured of NO˙ was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO˙ from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO˙ mg−1 protein min−1; was strictly dependent on NADPH, calmodulin, and BH4; and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO˙ could be involved in the process of senescence of pea leaves.

Metabolism of oxygen radicals in peroxisomes and cellular implications

Peroxisomes are subcellular respiratory organelles which contain catalase and H2O2-producing flavin oxidases as basic enzymatic constituents. These organelles have an essentially oxidative type of metabolism and have the potential to carry out different important metabolic pathways. In recent years the presence of different types of superoxide dismutase (SOD) have been demonstrated in peroxisomes from several plant species, and more recently the occurrence of SOD has been extended to peroxisomes from human and transformed yeast cells. A copper,zinc-containing SOD from plant peroxisomes has been purified and partially characterized. The production of hydroxyl and superoxide radicals has been studied in peroxisomes. There are two sites of O2- production in peroxisomes: (1) in the matrix, the generating system being xanthine oxidase; and (2) in peroxisomal membranes, dependent on reduced nicotinamide adenine dinucleotide (NADH), and the electron transport components of the peroxisomal membrane are possibly responsible. The generation of oxygen radicals in peroxisomes could have important effects on cellular metabolism. Diverse cellular implications of oxyradical metabolism in peroxisomes are discussed in relation to phenomena such as cell injury, peroxisomal genetic diseases, peroxisome proliferation and oxidative stress, metal and salt stress, catabolism of nucleic acids, senescence, and plant pathogenic processes.

Nitrosative stress in plants.

Nitrosative stress has become a usual term in the physiology of nitric oxide in mammalian systems. However, in plants there is much less information on this type of stress. Using olive leaves as experimental model, the effect of salinity on the potential induction of nitrosative stress was studied. The enzymatic l‐arginine‐dependent production of nitric oxide (NOS activity) was measured by ozone chemiluminiscence. The specific activity of NOS in olive leaves was 0.280 nmol NO mg−1 protein min−1, and was dependent on l‐arginine, NADPH and calcium. Salt stress (200 mM NaCl) caused an increase of the l‐arginine‐dependent production of nitric oxide (NO), total S‐nitrosothiols (RSNO) and number of proteins that underwent tyrosine nitration. Confocal laser scanning microscopy analysis using either specific fluorescent probes for NO and RSNO or antibodies to S‐nitrosoglutathione and 3‐nitrotyrosine, showed also a general increase of these reactive nitrogen species (RNS) mainly in the vascular tissue. Taken together, these findings show that in olive leaves salinity induces nitrosative stress, and vascular tissues could play an important role in the redistribution of NO‐derived molecules during nitrosative stress.

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.

A more sensitive modification of the catalase assay with the Clark oxygen electrode: Application to the kinetic study of the pea leaf enzyme

A modification of the method of catalase determination by means of the Clark oxygen electrode is described. The assay is based on measurement of the initial rate at which oxygen is released by catalase in an oxygen-free buffer. Displacement of oxygen was brought about by flushing with nitrogen, and the substrate used was hydrogen peroxide at a 33.5 mm final concentration. The method is rapid and can be used with crude catalase preparations. Its sensitivity is at least 20 times higher than that of previous methods; it has an interval of measurable activity of about 0.01–8.4 μmol of O2/min and, therefore, is applicable to an 840-fold range of catalase concentrations. This modification was applied to the kinetic study of crude extracts of pea leaf catalase. An apparent Km of 0.190 m was calculated.