Nitric Oxide Is Involved in Cadmium-Induced Programmed Cell Death in Arabidopsis Suspension Cultures

Exposure to cadmium (Cd²⁺) can result in cell death, but the molecular mechanisms of Cd²⁺ cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 μm CdCl₂ and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl₂ treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by N^G-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd²⁺-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd²⁺ cytotoxicity in plants.Cadmium (Cd²⁺) is a heavy metal with a long biological half-life, and its presence as a pollutant in agricultural soil is due mainly to anthropogenic activities. It is rapidly taken up by roots and enters the food chain, resulting in toxicity for both plants and animals (for review, see Sanità di Toppi and Gabbrielli, 1999). Cd²⁺ inhibits seed germination, decreases plant growth and photosynthesis, and impairs the distribution of nutrients. Overall, the symptoms of chronic exposure to sublethal amounts of Cd²⁺ mimic premature senescence (Rascio et al., 1993; McCarthy et al., 2001; Sandalio et al., 2001; Rodriguez-Serrano et al., 2006). Depending on the concentration, Cd²⁺ treatment of tobacco (Nicotiana tabacum) cell cultures and onion (Allium cepa) roots eventually triggers either necrosis or programmed cell death (PCD; Fojtovà and Kovařik, 2000; Behboodi and Samadi, 2004).Although Cd²⁺ is an environmental threat, the mechanisms by which it exerts its toxic effects in plants are not fully understood. In plant cells, Cd²⁺ is believed to enter through Fe²⁺, Ca²⁺, and Zn²⁺ transporters/channels (Clemens, 2006). Once in the cytosol, Cd²⁺ stimulates the production of phytochelatins (PCs), a glutathione-derived class of peptides containing repeated units of Glu and Cys, which bind the metal ions and transport them into the vacuole (Sanità di Toppi and Gabbrielli, 1999). Strong evidence exists that high (millimolar) concentrations of Cd²⁺ induce reactive oxygen species (ROS) bursts in plants, which might have a role in signaling and/or degenerative steps leading to cell death (Piqueras et al., 1999; Olmos et al., 2003; Cho and Seo, 2005; Garnier et al., 2006). Treatment with a lower, nontoxic Cd²⁺ concentration also caused increase in ROS production in pea (Pisum sativum) leaves and roots (Sandalio et al., 2001; Romero-Puertas et al., 2004; Rodriguez-Serrano et al., 2006) and Arabidopsis (Arabidopsis thaliana) cell cultures (Horemans et al., 2007).Nitric oxide (NO) is a gaseous reactive molecule with a pivotal signaling role in many developmental and response processes (for review, see Neill et al., 2003; Besson-Bard et al., 2008). In plants, it can be synthesized via several routes, either enzymatically or by chemical reduction of nitrite. Nitrate reductase and a root-specific plasma membrane nitrite-NO reductase also utilize nitrite as substrate. In animals, nitric oxide synthase (NOS) converts l-Arg into NO and l-citrulline. Although no plant NOS has been unambiguously identified yet, activity assays and pharmacological evidence suggests the existence of a NOS-like counterpart in plants. Depending on its concentration and possibly on the timing and localization of its production, NO can either act as an antioxidant or promote PCD, often in concert with ROS (Delledonne et al., 2001; Beligni et al., 2002; de Pinto et al., 2006). Extensive research has shown that NO plays a fundamental role in the hypersensitive response, but its involvement in other types of PCD, such as that resulting from mechanical stress and natural and cytokinin-induced senescence of cell cultures, has also been demonstrated (Garcês et al., 2001; Carimi et al., 2005). Because of its participation in numerous biotic and abiotic responses, NO has been proposed as a general stress molecule (Gould et al., 2003). However, the mechanisms by which NO determines its effects are far from being completely elucidated, and a number of downstream signaling pathways, involving Ca²⁺, cyclic GMP, and cyclic ADP-Rib, are involved (Neill et al., 2003; Besson-Bard et al., 2008). NO can also modulate biological responses by direct modification of proteins, reacting with Cys residues (S-nitrosylation), Tyr residues (nitration), or iron and zinc in metalloproteins (metal nitrosylation; Besson-Bard et al., 2008).The aim of this work is to study the plant responses to various concentrations of Cd²⁺ and, in particular, the role of ROS and NO in the signaling events leading to cell death. Cell cultures of the model plant Arabidopsis were chosen as an experimental system because the homogeneity and undifferentiated state of the cells, combined with the uniform delivery of the treatments, allow a clear and reproducible response. The results point to NO as a master regulator of Cd²⁺-induced cell death. Possible mechanisms that explain this evidence will be discussed.