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 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 À1 protein min À1 , and was dependent on L L-arginine, NADPH and calcium. Salt stress (200 mM NaCl) caused an increase of the L 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.

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

Post-translational modifications (PTMs) mediated by nitric oxide (NO)-derived molecules have become a new area of research, as they can modulate the function of target proteins. Proteomic data have shown that ascorbate peroxidase (APX) is one of the potential targets of PTMs mediated by NO-derived molecules. Using recombinant pea cytosolic APX, the impact of peroxynitrite (ONOO–) and S-nitrosoglutathione (GSNO), which are known to mediate protein nitration and S-nitrosylation processes, respectively, was analysed. While peroxynitrite inhibits APX activity, GSNO enhances its enzymatic activity. Mass spectrometric analysis of the nitrated APX enabled the determination that Tyr5 and Tyr235 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Residue Cys32 was identified by the biotin switch method as S-nitrosylated. The location of these residues on the structure of pea APX reveals that Tyr235 is found at the bottom of the pocket where the haem group is enclosed, whereas Cys32 is at the ascorbate binding site. Pea plants grown under saline (150mM NaCl) stress showed an enhancement of both APX activity and S-nitrosylated APX, as well as an increase of H2O2, NO, and S-nitrosothiol (SNO) content that can justify the induction of the APX activity. The results provide new insight into the molecular mechanism of the regulation of APX which can be both inactivated by irreversible nitration and activated by reversible S-nitrosylation.

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.