RESULTATS AND DISCUSSION International Symposium Glutathione and related thiols in microorganisms and plants, Faculté de Pharmacie de Nancy 26-29 August 2008 EFFECT OF CADMIUM ON GLUTAREDOXINS OF PEA (Pisum sativum L.) DURING GERMINATION SMIRI M.1,2, CHAOUI A.1, ROUHIER N.2, CHIBANI K.2, JACQUOT JP.2 and EL FERJANI E.1 1-Bio-Physiologie Cellulaires, Faculté des Sciences de Bizerte, 7021-Zarzouna (Tunisie). 2-Unité Mixte de Recherche, 1136 Interaction arbres microorganismes INRA, Université Henri-Poincaré, Faculté des Sciences, BP 239, 54506 Vandoeuvre cedex (France). E mail: Moez.Smiri@scbiol.uhp-nancy.fr INTRODUCTION Heavy metals have a negative impact on seed germination and they are believed to interfere with the enzymatic activities of redox proteins. More specifically, so far little is known about the behaviour of redox enzymes in seeds under Cd stress and the mechanisms linked to the effects of heavy metal contamination are still unclear. We report here the response of glutaredoxins in cotyledons and embryonic axis of germinating pea seeds exposed to a treatment with a toxic Cd concentration. The glutaredoxin activities and the glutaredoxin protein contents have been analysed in these stress conditions. MATERIALS AND METHODS Seeds of pea (Pisum sativum L. cv. douce province) were disinfected with 2% of sodium hypochlorite for 10 min and then rinsed thoroughly and soaked in distilled water at 4 °C for 30 min to obtain an initial stage. Seeds were germinated at 25 °C in the dark for five days over two sheets of filter paper moistened with distilled water or an aqueous solution of 5 mM CdCl2. At harvest, coats were removed and the embryonic axis and cotyledons weighed and stored at -20 °C until use. Cotyledons and embryonic axis were homogenized with 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM MgCl2 and 14 mM β-mercaptoethanol (1:5 [w/v]). After 25 min of centrifugation at 15,000 g, the supernatant was collected and its protein concentration determined with the Bradford assay, with bovine serum albumin as the standard (Bradford, 1976, [Anal Biochem 72: 248–254]). Electrophoresis in polyacrylamide gel was performed according to Laemmli (1970), [Nature 227: 680–685], proteins were transferred to a PVDF membrane, and western blotting was performed as in Towbin et al. (1979), [Proc Natl Acad Sci USA 76: 4350–4354], using poplar Grx C4 antibody. Glutaredoxin activity was determined by a coupled enzymatic reaction (Holmgren,A. and Åslund,F. 1995, [Methods Enzymol 252: 199-208]). The NADPH-dependent reduction of 2-hydroxyethyl disulfide was followed at 340 nm in the presence of glutathione reductase and cotyledons or embryonic axis extract containing protein as the source of glutaredoxin activity. The assay mixture contained 30 mM Tris–HCl, pH 8.0, 1 mM EDTA, 0.2 mM NADPH, 6 µg/mL glutathione reductase, 0.5 mM GSH and 0.5 mM 2-hydroxyethyl disulfide in a total volume of 500 µl. RESULTATS AND DISCUSSION Total protein content A B Glutaredoxin activities A B Figure 1 : Total protein content in cotyledons (A) and embryonic axis (B) of pea seeds during germination after imbibition with H2O or 5 mM Cd. The values represent the average of 6 individual measurements (±SE). Each measurement was performed in an extract obtained from several germinating seeds. SDS-PAGE B A Figure 4 : Enzymatic capacities of glutaredoxins in cotyledons (A) and embryonic axis (B) of pea seeds during germination after imbibition with H2O or 5 mM Cd. The values represent the average of 6 individual measurements (±SE). Each measurement was performed in an extract obtained from several germinating seeds. Figure 2 : 10 µg Soluble proteins from cotyledons (A) and embryonic axis (B) of pea seeds imbibed for 0 to 120 h with H2O or 5 mM Cd were loaded per lane. Figure 3 : 20 µg Soluble proteins from cotyledons (A) and embryonic axis (B) of pea seeds imbibed for 0 to 120 h with H2O or 5 mM Cd were loaded per lane. Proteins were then transferred onto PVDF membranes that were probed with poplar anti- Grx C4 . Line 1, poplar recombinant Grx C4. CONCLUSIONS Our data suggest that glutaredoxins plays an important role during pea seed germination in the presence of high Cd concentrations : An increase in total protein amounts was observed in cotyledons and embryonic axis during heavy metal poisoning (Fig 1). In embryonic axis, the amount of Grx C4 does not change in presence or absence of cadmium, while Grx C4 abundance is increased under Cd treatment in cotyledons between 24 and 120 h (Fig 3). Cd treatment maintained glutaredoxin activities at a higher level than that in control seeds (Fig 4).