Signal transduction pathways during cadmium stress in sunflower discs

M.P. IANUZZO; M.D. GROPPA; M.P. BENAVIDES; M.L. TOMARO

Buenos Aires, Argentina

Congreso; Baires Biotec 2005. Congreso internacional. Grupo Biotecnología. VI Simposio Nacional de Biotecnología. REDBIO Argentina 2005. Encuentro trinacional REDBIO Argentina-Chile-Uruguay; 2005

Introduction The reactive oxygen species (ROS) have been considered for many years an unavoidable consequence of living in an aerobic environment. But in recent years, accumulated evidence suggests that ROS such as superoxide anion (O2.-) and H2O2, function also as intracellular second messengers. Cadmium is a heavy metal that produce oxidative stress by still not completely clarified mechanisms. The objective of the present work was to elucidate which type of ROS mediates the signaling pathways involved in cadmium toxicity in sunflower leaves, by using several inhibitors in order to confirm the involvement of different enzymatic systems in ROS formation as well as the possible implication of O2.-/H2O2 in the signal transduction pathway during Cd2+ stress. Materials and Methods Sunflower seeds were germinated and grown in a growth chamber, under a photoperiod of 16/8 h, at 23 ± 2 °C, for 20 days, watered with Hoagland solution. The first pair of leaves was used to obtain the discs that were used for the assays. Leaf discs were cut with a cork borer and preincubated with different inhibitors or inducers (100 mM DPI, 1 mM LaCl3, 10mM Cl2Ca, 100 mM ODQ, 1 mM EGTA, 0.5 mM Cl2Zn) for 3 h under continuous illumination, and then transferred to either distilled water or 0.5 mM Cl2Cd for another 14h. After the incubation period, discs were either inmersed in diaminobenzidine (DAB) or nitrobluetetrazolium (NBT) solutions to localize H2O2 and O2.- production, respectively. Extracts were made to quantify thiobarbituric acid reactive substances (TBARS), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) activities. Results and Discussion TBARS content In order to evaluate the oxidative damage at the level of plasma membranes, TBARS formation was measured, in the tissues and in the incubation medium. Cd elevated TBARS content an average of 30% over the controls, and this increase was not significantly reverted by any of the inhibitors tested. This results seems to indicate that signals that lead to induce damage at the level of membranes was not directly related to ROS generation. Total GSH content Total GSH was measured as a soluble marker of oxidative damage (Fig.1). The metal markedly reduced GSH content to 24% of the control value. The blockage of Ca2+ influx by LaCl3 almost completely returned GSH content to the level of controls. This result suggests that GSH depletion is Ca2+-dependent. However, Ca2+ only partially reversed GSH depletion, while ODQ, a cGMP inhibitor, induced a recovery of 50% in GSH content respect to the Cd2+-induced GSH reduction. DPI, a NADPH oxidase inhibitor, and EGTA by themselves reduced the thiol content. Zn2+, used as a membrane protector, reverted Cd-induced GSH-depletion almost 50% respect to the control. NBT reduction assay The production of O2.- was studied by infiltrating leaves with NBT, which is reduced by O2.-, producing dark spots of blue formazan. In the first assay, 10, 100 and 500 µM Cd2+ were used, and samples were taken at 1, 3, 6 and 14 h of treatment (Fig. 2). Cd2+, at 100 and 500 µM, partially inhibited O2.- formation at 3h of incubation, while a clear inhibition was observed at 6 h. The lowest metal concentration (10 µM) inhibited the radical formation only after 6 h of Cd exposure. Inhibitors were tested only when leaf discs were treated for 14 h. DPI and EGTA completely inhibited O2.- formation in control discs. LaCl3 reversed Cd2+-induced inhibition of O2.- formation, but ODQ, Ca2+ and Zn2+ did not. For that reason, it seemed evident that O2.- formation inhibited by Cd2+ was not related to cGMP or Ca2+ exogenous supply, but might be depending on Ca2+ continuous influx from extracellular medium through specific channels at the plasma membrane. However, LaCl3 could also be blocking Cd2+ influx through Ca2+ channels(cita). DAB  assay Leaf discs were infiltrated with DAB to evidence, in situ, the accumulation of H2O2, revealed as brown spots. The first assay was conducted as with NBT. The presence of H2O2 was increasing with time and Cd2+ concentration. At 14 h of incubation, DPI and EGTA completely abolished H2O2 precipitation. The results obtained trying to evidence H2O2 formation using different inhibitors or effectors was not clear enough to definitively assessed their involvement in Cd2+-induced ROS evolution SOD and CAT activity A very slight increase in SOD activity was observed in leaf discs under Cd2+ stress and this result was not modify by none of the inhibitors tested. CAT activity decreased in discs under stress, while LaCl3 and Ca2+ partially reversed this decay, showing the involvement of Ca2+ influx in ROS evolution under Cd2+ treatment.           Fig 1. GSH content in sunflower leaves   Fig. 2. O2.- formation in sunflower discs Conclusions The results obtained showed evidence about a time and concentration–dependent inhibition of O2.- accumulation in Cd2+-treated sunflower leaves. This result seemed to be more a consequence of NADPH like-enzyme inhibition by Cd2+ than a result of an increase of SOD activity. In an in vitro assay, NADPH oxidase-like activity was inhibited by Cd2+ in a dose-dependent manner (data not shown). Calcium could be involved in the signal transduction pathway that lead to the inhibition of O2.-formation by Cd2+ in sunflower leaves, regarding the effect exerted by a Ca2+ channel blocker LaCl3. Other assays are currently under investigation to elucidate this point.