Final Report Summary - NO Signal (S-nitrosylation mediated signalling during the response to different stresses: A proteomic-based approach?) Peroxisomes are organelles that have an essentially oxidative type of metabolism (Huang et al. 1983; Fahimi and Sies 1987; Baker and Graham 2002) and the important role of plant peroxisomes in a variety of metabolic reactions such as photorespiration, fatty acid ß-oxidation and the glyoxylate cycle is well known (del Rio et al., 2009). Peroxisomes are also essential in plant responses to abiotic and biotic stresses (Romero-Puertas et al., 1999; Koh et al., 2005) increasing even their population during different stress conditions such as xenobiotics, ozone, cadmium, H2O2 and light (del Rio et al., 2009). In the last two decades, in plant peroxisomes it has been shown the production of reactive oxygen species (ROS) such as, hydrogen peroxide (H2O2) and radical superoxide (O2.-) as a consequence of their normal metabolism (see del Río et al., 2009). In addition, a complex battery of antioxidative systems has been described, including catalase (CAT), superoxide dismutases (SODs; see del Río et al. 2002a) and the ascorbate-glutahione cycle (Jiménez et al. 1997; Mittova et al 2004; Kuzniak and Sklodowska, 2005; Romero-Puertas et al 2006). Recently, the existence of an L-arginine-dependent nitric oxide synthase (NOS) and the presence of nitric oxide (NO) in plant peroxisomes have been shown as well (Barroso et al 1999; Corpas et al 2004). In plants, NO is a key signalling molecule involved in several physiological processes from development to defence responses to both, biotic and abiotic stress (Delledonne 2005). The occurrence of NO in peroxisomes adds new cellular functions to these organelles related to oxygen and nitrogen reactive species (ROS/RNS). No function for NO in peroxisomes, however, has been described so far. The way by which NO devise the plethora of physiological functions where it is involved is still largely unknown. For a long time in animal tissues and very recently in vegetal tissues it has been shown that NO regulates diverse biologic processes by directly modifying proteins. Actually, NO and RNS can oxidize, nitrate or nitrosylate proteins. S-nitrosylation refers to the binding of a NO group to a cysteine residue and it plays a significant role in NO-mediated signalling (Stamler, et al., 2001). Very recent evidences suggest that many proteins are S-nitrosylated in plants under physiological or stress conditions (Lindermayr et al., 2005; Romero-Puertas et al.,2008), and the first insights into the SNO-dependent regulation of protein function has been obtained (Lindermayret al., 2006; Romero-Puertas et al., 2007; Wang et al., 2009). S-nitrosylation leads not only to changes in protein activity but in protein-protein interactions and/or subcellular localization (Benhar et al., 2005) and therefore spatiotemporal distribution of SNO-proteins is a key component of S-nitrosylation significance. Several peroxisomal proteins has been shown to be regulated by NO, such us glutathione peroxidase that are down-regulated by NO, whereas the activity of the peroxisomal H2O2-producing ß-oxidation is enhanced by NO. Furthermore, NO inhibits, in a reversible way, the activities of tobacco catalase and ascorbate peroxidase, whereas both of them are irreversibly inhibited by peroxynitrite. Foster and Stamler (2004) have shown a few mitochondrial proteins as well as catalase to be S-nitrosylated in rat liver and very recently Palmieri et al. (2010) identified 11 candidate that were S-nitrosylated in mitochondria of Arabidopsis leaves, suggesting that protein S-nitrosylation in subcellular organelles, might be precisely regulated and possibly in a reversible way. During the execution of this project we have assessed for the first time the occurrence of S-nitrosylation in plant peroxisomes (Objective 1). We have identified 6 peroxisomal proteins as target for S-nitrosylation which belong to peroxisomal metabolism such us ROS detoxification, ß-oxidation and photorespiration using proteomic techniques involving the specific biotin-switch method for detection and purification of S-nitrosylated proteins and two different mass spectrometry techniques (MALDI-TOF/TOF and 2DnLC-MS/MS). Additionally, S-nitrosylation impact on functionality of three key peroxisomal proteins, such as catalase (CAT), glycolate oxidase (GOX) and malato dehydrogenase (MDH), and their pattern of S-nitrosylation under two different abiotic stress conditions, such us cadmium and 2,4-D have been analyzed (Objective 1 and 2). So, we could observe a significant inhibition of these enzyme activities after GSNO incubation in a concentration-dependent manner, up to 25-30 % when treated with 1 mM GSNO. Moreover, CAT and GOX were found to be S-nitrosylated under physiological conditions. A reduction of S-nitrosylated CAT under Cd treatment while no differences under 2,4-D treatment with respect to the non treated plants was observed. S-nitrosylated GOX was however strongly reduced under both abiotic stresses. Finally, we have identified the S-nitrosoproteome of mitochondria and its changes under salt stress (Objective 3). These results has allowed us to identified a new mechanism (S-nitrosylation) that regulate the peroxisomal and mitochondrial level of key signalling molecules, such us ROS and RNS, and have supplied more information on these organelles contribution throughout plant development and in plant response to different stress condition. Plants are continually exposed to a wide array of potential abiotic stresses and have evolved a battery of protective disease resistance mechanisms. Although disease is the exception rather than the rule, when protective mechanisms break down the consequences can be devastating with major crop losses pre- and post-harvest. It is anticipated that advances in SNO biology may provide novel opportunities for both rational crop design and plant breeding potentially to improve a plethora of traits relevant to agriculture. Bibliography: -Baker A , Graham I (2002) Plant peroxisomes. Biochemistry, cell biology and biotechnological applications . 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