| Abstract: | Peroxisomes are unique organelles involved in multiple cellular metabolic pathways. Nitric oxide (NO) is a free radical active in many physiological functions under normal and stress conditions. Using Arabidopsis (Arabidopsis thaliana) wild type and mutants expressing green fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo, this study analyzes the temporal and cell distribution of NO during the development of 3-, 5-, 8-, and 11-d-old Arabidopsis seedlings and shows that Arabidopsis peroxisomes accumulate NO in vivo. Pharmacological analyses using nitric oxide synthase (NOS) inhibitors detected the presence of putative calcium-dependent NOS activity. Furthermore, peroxins Pex12 and Pex13 appear to be involved in transporting the putative NOS protein to peroxisomes, since pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, registered lower NO content. Additionally, we show that under salinity stress (100 mm NaCl), peroxisomes are required for NO accumulation in the cytosol, thereby participating in the generation of peroxynitrite (ONOO−) and in increasing protein tyrosine nitration, which is a marker of nitrosative stress.Peroxisomes are single membrane-bound organelles whose basic enzymatic constituents are catalase and H2O2-producing flavin oxidases as their basic enzymatic and are found in virtually all eukaryotic cell types (Corpas et al., 2001; Hayashi and Nishimura, 2006; Reumann et al., 2007; Pracharoenwattana and Smith, 2008; Palma et al., 2009). These oxidative organelles are characterized by metabolic plasticity, as their enzymatic content can vary according to the organism, cell/tissue type, and environmental conditions (Mullen et al., 2001; Hayashi and Nishimura, 2003; Corpas et al., 2009a). In higher plants, peroxisomes contain a complex battery of antioxidative enzymes, such as catalase, superoxide dismutase, the components of the ascorbate-glutathione cycle, and the NADP-dehydrogenases of the pentose-P pathway (Corpas et al., 2009a). The generation of superoxide radicals has also been reported in the matrices and membranes of peroxisomes (López-Huertas et al., 1999; del Río et al., 2006). All these findings point to the important role played by peroxisomes in the cellular metabolism of reactive oxygen species (Corpas et al., 2001, 2009a; del Río et al., 2006).Nitric oxide (NO) is a free radical involved in many physiological functions under normal and stress conditions in both animal and plant cells (Arasimowicz and Floryszak-Wieczorek, 2007; Corpas et al., 2007a, 2008; Neill et al., 2008). Unlike animal systems, knowledge of NO generation and subcellular location in plants remains largely elusive, and the data are sometimes contradictory and ambiguous (Zemojtel et al., 2006; Jasid et al., 2006; Gas et al., 2009). In previous studies, we detected l-Arg-dependent nitric oxide synthase (NOS) activity in isolated pea (Pisum sativum) leaf peroxisomes (Barroso et al., 1999). In a later study, using electron paramagnetic resonance techniques, we demonstrated the presence of NO in these types of peroxisomes (Corpas et al., 2004). However, several issues, such as whether NO is released into the cytosol and the physiological function of this free radical, remain unresolved.In this study, we provide an in vivo demonstration that Arabidopsis peroxisomes are essential for NO accumulation in the cytosol, thus participating in the generation of nitrosative stress under salinity conditions. In addition, using Arabidopsis mutants pex12 and pex13, we also suggest that these peroxins are involved in importing into peroxisomes the enzyme responsible for NO generation. |
