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. ) is a widespread intracellular and intercellular messenger with a broad spectrum of regulatory functions in many physiological processes (Moncada et al., 1991;Ignarro, 2002;Wendehenne et al., 2001;Lamattina et al., 2003;Neill et al., 2003;del Río et al., 2004). In recent years, NO. was reported to be involved in many key physiological processes of plants, such as ethylene emission (Leshem and Haramaty, 1996), response to drought (Leshem, 1996), disease resistance (Delledonne et al., 1998(Delledonne et al., , 2001Durner et al., 1998;Clarke et al., 2000), growth and cell proliferation (Ribeiro et al., 1999), maturation and senescence (Leshem et al., 1998), apoptosis/programmed cell death (Magalhaes et al., 1999;Clarke et al., 2000;Pedroso and Durzan, 2000;Pedroso et al., 2000a;Zhang et al., 2003), and stomatal closure Lamattina, 2001, 2002;Neill et al., 2002a;García-Mata et al., 2003).The application of exogenous NO . to plants has been used as a tool to study how this molecule affects some physiological processes, such as inhibition of certain enzyme activities (Clark et al., 2000;Navarre et al., 2000), cell wall lignification (Ferrer and Ros Barceló , 1999) In animal systems, a considerable attention is being dedicated to this molecule and the enzyme responsible for its production from L-Arg, nitric oxide synthase (NOS; EC 1.14. 13.39;Hemmens and Mayer, 1998; Alderton et al., 2001). On the contrary, in plants comparatively much less is known on the source of NO . production (Neill et al., 2003;Wendehenne et al., 2003;del Río et al., 2004 Article, publication date, and citation information can be found at www.plantphysiol.org/cgi

In pea ( Pisum sativum L.) leaves from plants grown in the presence of 50 µ µ µ µ M CdCl 2 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 H 2 O 2 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 H 2 O 2 , 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.

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.

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 Ca

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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 (NO2-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.