Potential role of NADPH-oxidase in early steps of lead-induced oxidative burst in Vicia faba roots

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Summary

The mechanism of oxidative burst induced by lead in Vicia faba excised roots was investigated by luminol-dependent chemiluminescence. Results showed that lead triggered a rapid and dose-dependent increase in chemiluminescence production. In this study, specific inhibitors of putative reactive oxygen species (ROS) sources were used to determine the mechanism of lead-induced ROS generation. This generation was sensitive to dephenylene iodonium (DPI), quinacrine and imidazole, some inhibitors of the NADPH-oxidase and not inhibited by other putative ROS sources inhibitors. Data reported in this work clearly demonstrated the pivotal role of NADPH-oxidase-like enzyme in early steps of lead-induced oxidative burst. To investigate the respective implication of calmodulin and protein kinase (PK) in lead-induced NADPH-oxidase activation, excised roots were treated with the calmodulin inhibitor W7 or with the PK inhibitor staurosporine. The chemiluminescence generation inhibition by these inhibitors illustrated the role of PK in lead-induced NADPH-oxidase activation and revealed a calmodulin-dependent step. Using the calcium entry blocker La3+ or different concentrations of calcium in the extra-cellular medium, our data highlighted the implication of Ca2+ channel in lead-induced oxidative burst.

Introduction

Lead is one of the most abundant heavy metals polluting the soil and environment. It originates from numerous sources and has become an environmental concern (Singh et al., 1997). Lead is toxic to many organs: it is a proven animal carcinogen (Johnson, 1998) and an inducer of many toxic symptoms in plants, such as decrease of growth due to interference with enzymes essential for normal metabolism and development, photosynthetic processes, water and mineral nutrient absorption and changes in cell ultrastructure (Singh et al., 1997). Lead is reported to produce reactive oxygen species (ROS) and to alter the activities of antioxidant enzymes (Malecka et al., 2001; Verma and Dubey, 2003). These ROS include superoxide anions (O2radical dot), hydroxyl radical (OHradical dot), singlet oxygen (1O2) and hydrogen peroxide (H2O2), which are produced during membrane-linked electron transport activities as well as by a number of metabolic pathways (Shah et al., 2001).

The rapid production of ROS, or “oxidative burst”, was originally identified in mammalian phagocytes and later demonstrated in plant cells (Low and Merida, 1996). It is believed that, in plants, putative sources of ROS during biotic or abiotic stresses are cell-wall-bound peroxidases (Kawano, 2003), xanthine oxidase, amine oxidases (AOs) in the apoplast (Allan and Fluhr, 1997) and plasma-membrane-bound NADPH-oxidases (Bolwell and Wojtaszek, 1997). The key enzyme that contributes to ROS formation in phagocytes is a plasma membrane-bound NADPH-dependent oxidase (Segal and Abo, 1993), the activation of which involves the assembly of at least three cytosolic proteins (p67phox, p47phox and p40phox), two plasma membrane-associated ones (gp91phox and p22phox) and two small GTP-binding proteins (p21rac and Rap1A). The activation of this complex induces the translocation and the association of the cytoplasmic proteins to the membrane-bound components. This process involves the phosphorylation of at least p47phox by protein kinase C (PKC). Pharmacological approaches with dephenylene iodonium (DPI) demonstrate the involvement of a NADPH-oxidase-like enzyme in elicited cells of Arabidopsis thaliana (Desikan et al., 1996). Moreover, immunological studies using mammalian NADPH-oxidase subunit antibodies have reported the presence of subunits of this enzyme complex (Desikan et al., 1996; Razem and Bernards, 2003). Recently, Overmyer et al. (2003) identified respiratory burst oxidase homologues as plant homologues of the catalytic subunit of phagocyte NADPH-oxidase (gp91phox) that were reported to be responsible for ROS production during biotic stress (Simon-Plas et al., 2002). Cadmium-induced oxidative stress is also thought to be mediated by an NADPH-oxidase-like enzyme (Garnier et al., 2006; Olmos et al., 2003). Garnier et al. (2006) suggested that cadmium enters cells via La3+-sensitive transporters or calcium channels to generate this oxidative burst. Lead entry into plant cells also occurs, at least in part, through Ca2+-permeable channels (Huang and Cunningham, 1996; Sunkar et al., 2000).

Although lead toxicity appears related to oxidative stress (Malecka et al., 2001; Verma and Dubey, 2003), the mechanism leading to ROS production remains unclear. An attempt was made in this study to determine the effect of lead on ROS production by chemiluminescence (CL), in lead-treated Vicia faba (Vf) roots. Specific inhibitors of putative ROS sources were used to determine the mechanism at the origin of lead-induced ROS generation and the role of NADPH-oxidase-like enzyme.

Section snippets

Plant material and growth

Broad beans (Vf, L.) were germinated on filter paper moistened with deionized water at 25 °C in the dark. After 7 d, plants were grown in a PVC tank (four plants per tank) containing 500 mL of aerated Hoagland solution. To keep the nutrient composition and pH constant, solutions were renewed daily. The culture systems were located in a growth chamber with day/night air temperatures of 24/22 °C and relative humidity of 70/75%. Plants were provided light 16 h a day. A 600 W Osram Nav-T Super High

Induction of oxidative burst by lead

Induction of Vf excised roots CL activity was measured in the presence of luminol as enhancer, for 120 s at 25 °C after lead treatment (Figure 1A). Lead stimulation was carried out under continuous CL recording. In the absence of lead, the time course of CL production remained constant at a low level (0.45±0.05 mV). In the presence of lead, after a lag time of 15±3 s, CL production increased rapidly and reached a maximum value after 90±15 s of lead stimulation. The CL decreased slowly to the basal

Discussion

In this study, oxidative burst was investigated by luminol-dependent-CL. In excised Vf roots, lead triggered a rapid and dose-dependent increase in ROS production (Figure 1A and B). Results reported in Figure 2 suggested that, at least in first step, lead-induced oxidative burst is mediated by O2radical dot and not by H2O2. In plants, four biological systems are putative sources of ROS during biotic or abiotic stresses: plasma-membrane-bound NADPH-oxidases, cell-wall-bound peroxidases, AOs in the

Conclusion

In summary, our results suggest that lead entry in root cells may occur via calcium-permeable channels. Data also demonstrated that the early step in lead-induced oxidative burst in Vf roots is strongly associated with NADPH-oxidase activation. This study underlined the importance of a PK in NADPH-oxidase-like enzyme activation and revealed a calmodulin-dependent step in lead-induced ROS production. However, NADPH-oxidase may not be the only source of ROS after hours of exposure. Garnier et al.

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