Cellular response of pea plants to cadmium toxicity: Cross talk between reactive oxygen species, nitric oxide, and calcium

Abstract

Cadmium (Cd) toxicity has been widely studied in different plant species; however, the mechanism involved in its toxicity as well as the cell response against the metal have not been well established. In this work, using pea (Pisum sativum) plants, we studied the effect of Cd on antioxidants, reactive oxygen species (ROS), and nitric oxide (NO) metabolism of leaves using different cellular, molecular, and biochemical approaches. The growth of pea plants with 50 μm CdCl2 affected differentially the expression of superoxide dismutase (SOD) isozymes at both transcriptional and posttranscriptional levels, giving rise to a SOD activity reduction. The copper/zinc-SOD down-regulation was apparently due to the calcium (Ca) deficiency induced by the heavy metal. In these circumstances, the overproduction of the ROS hydrogen peroxide and superoxide could be observed in vivo by confocal laser microscopy, mainly associated with vascular tissue, epidermis, and mesophyll cells, and the production of superoxide radicals was prevented by exogenous Ca. On the other hand, the NO synthase-dependent NO production was strongly depressed by Cd, and treatment with Ca prevented this effect. Under these conditions, the pathogen-related proteins PrP4A and chitinase and the heat shock protein 71.2, were up-regulated, probably to protect cells against damages induced by Cd. The regulation of these proteins could be mediated by jasmonic acid and ethylene, whose contents increased by Cd treatment. A model is proposed for the cellular response to long-term Cd exposure consisting of cross talk between Ca, ROS, and NO. Cadmium (Cd) is a toxic element whose presence in the environment is mainly due to industrial processes and phosphate fertilizers and then is transferred to the food chain (Pinto et al., 2004). Cd is rapidly taken up by plant roots and can be loaded into the xylem for its transport into leaves. Most plants are sensitive to low Cd concentrations, which inhibit plant growth as a consequence of alterations in the photosynthesis rate and the uptake and distribution of macronutrients and micronutrients (Lozano-Rodriguez et al., 1997; Sandalio et al., 2001; Benavides et al., 2005). It is known that the content of polyvalent cations can be affected by the presence of Cd through competition for binding sites of proteins or transporters (Gussarson et al., 1996). Thus, Cd produced a decrease of calcium (Ca) content in different plant species (Gussarson et al., 1996; Sandalio et al., 2001). Ca is involved in the regulation of plant cell metabolism and signal transduction (Yang and Poovaiah, 2002; Rentel and Knight, 2004) and modulates cellular processes by binding proteins such as calmodulin (CaM), which in turn regulates the activity of target proteins (Roberts and Harmon, 1993).

Cd can be detoxified by phytochelatins, whose synthesis is induced by Cd and other metals and is accompanied by a decrease in the concentration of glutathione (Zenk, 1996). In addition, Cd produces disturbances in the plant antioxidant defenses, producing an oxidative stress (Somashekaraiah et al., 1992; Shaw, 1995; Gallego et al., 1996; Dixit et al., 2001; Sandalio et al., 2001; Schützendübel et al., 2001; Romero-Puertas et al., 2002, 2007; Rodríguez-Serrano et al., 2006). Recently, the cellular production of reactive oxygen species (ROS) in leaves from pea (Pisum sativum) plants under Cd stress has been reported (Romero-Puertas et al., 2004). ROS were detected in epidermal, transfer and mesophyll cells, with plasma membrane being the main source of ROS, although mitochondria and peroxisomes were also involved (Romero-Puertas et al., 2004). Concerning the mechanism of ROS production, Cd does not participate in Fenton-type reactions (Stoch and Bagchi, 1995) but can indirectly favor the production of different ROS, such as hydrogen peroxide (H2O2), superoxide (O2·−), and hydroxyl radical (·OH), by unknown mechanisms, giving rise to an oxidative burst (Olmos et al., 2003; Romero-Puertas et al., 2004; Garnier et al., 2006). The enzymes superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) are involved in the detoxification of O2·− (SOD) and H2O2 (CAT, POX), thereby preventing the formation of ·OH radicals. Ascorbate peroxidase and glutathione reductase, as well as glutathione, are important components of the ascorbate-glutathione cycle responsible for the removal of H2O2 in different cellular compartments (Jiménez et al., 1997; Noctor et al., 1998). Apart from their toxic roles, ROS are also involved in signaling in different processes such as growth, development, and response to biotic and abiotic stresses, and this signaling process is controlled by a balance between ROS production and scavenging (Bailey-Serres and Mittler, 2006).

Nitric oxide (NO) is a widespread intracellular and intercellular messenger with a broad spectrum of regulatory functions in many physiological processes (Moncada et al., 1991; Ignarro, 2002; del Río et al., 2004; Grün et al., 2006). In plants, NO was reported to be involved in ethylene (ET) emission (Leshem and Haramaty, 1996), response to drought (Leshem, 1996), disease resistance (Durner et al., 1998; Clark et al., 2000; Delledonne et al., 2001), growth and cell proliferation (Ribeiro et al., 1999), maturation and senescence (Leshem and Haramaty, 1996; Corpas et al., 2004), apoptosis/programmed cell death (Magalhaes et al., 1999; Clark et al., 2000; Pedroso and Durzan, 2000), and stomatal closure (García-Mata and Lamattina, 2002; Neill et al., 2002). There are several enzymatic systems that have been shown to produce NO, mainly nitrate reductase (Rockel et al., 2002) and l-Arg-dependent nitric oxide synthase (NOS; Corpas et al., 2004). However, the gene for the plant NOS has not been identified yet (Zemojtel et al., 2006; Neill et al., 2008).

In this work, the effect of growing pea plants with CdCl2 on the production of ROS and NO in leaves was studied in vivo by confocal laser microscopy. Taking into account that in pea plants the NOS-derived NO production is dependent on Ca (Corpas et al., 2004), the effect of this metal on NO and ROS production as well as the SOD activity were also investigated. To get deeper insights into the mechanisms of cellular response to Cd toxicity, the roles of different molecules that could be involved in cell signaling under metal stress, such as jasmonic acid (JA) and salicylic acid (SA), as well as ET, were studied. In addition, the expression of the antioxidative enzymes SOD, pathogen-related proteins (PRs), and defense-related proteins have been analyzed. All of these pieces of information are very important to understanding the mechanisms involved in the defense of plant cells against different types of abiotic stress