Cadmium and H2O2-induced oxidative stress in Populus × canescens roots

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Abstract

Clonal, hydroponically grown poplar plants (Populus × canescens, a hybrid of Populus tremula × Populus alba) were exposed to Cd or H2O2 to find out whether Cd-induced injury was related to the disturbance of the cellular redox control in root tips. Cd exposure resulted in an inhibition of antioxidative enzymes (superoxide dismutase, EC 1.15.1.1; catalase, EC 1.11.1.6; ascorbate peroxidase, EC 1.11.1.11; monodehydroascorbate radical reductase, EC 1.1.5.4; glutathione reductase, EC 1.6.4.2) but had fewer effects on dehydroascorbate reductase (EC 1.8.5.1) activities. Glutathione concentrations decreased, whereas ascorbate remained unaffected by Cd. Five micromoles of Cd were subinjurious in short-term experiments and stimulated root growth. Fifty micromoles of Cd retarded shoot growth faster than root growth, caused a more severe loss in antioxidative capacity than 5 μM Cd and resulted in an accumulation of H2O2 in roots. Exposure to H2O2 had an effect on antioxidative enzymes similar to that found under the influence of Cd, but caused GSH accumulation, and loss of ascorbate. The present data indicate that both agents acted via the disturbance of the cellular redox control.

Introduction

Cadmium (Cd) is highly toxic to animals and plants. In plants, exposure to Cd causes reductions in photosynthesis, water and nutrient uptake 〚29〛. As a consequence Cd-exposed plants show various symptoms of injury such as chlorosis, growth inhibition, browning of root tips, and finally death 〚15〛. Cd pollution of soils is of growing concern because Cd and other heavy metals are emitted by traffic, metalworking industries, mining, and as a by-product of mineral fertilisers 〚22〛. In addition to anthropogenic emissions, Cd is also released into the environment by natural sources such as volcanoes and continental dusts or by weathering of the underlying bedrock. Since the presence of Cd or other heavy metals prevents the development of a normal vegetation cover, biotechnological efforts are underway to develop more stress-tolerant species. For this purpose, it is important to understand the mechanisms of Cd toxicity and tolerance in plants.

It is well known that exposure of plants to Cd induces the synthesis of phytochelatins 〚27〛, 〚39〛. These peptides bind heavy metals in the cytosol and sequester them in the vacuole 〚18〛, 〚27〛. The tripeptide glutathione (γ-glutamylcysteinylglycine) is thought to play a central role in the Cd detoxification because it is the precursor for the synthesis of phytochelatins. The degree of Cd tolerance was correlated with inherent glutathione levels and the cellular capacity to synthesise thiol compounds 〚14〛, 〚35〛, 〚38〛, 〚40〛, 〚41〛. In hybrid poplar (P. × canescens, a hybrid of P. tremula × alba) with modified GSH concentrations, increased Cd accumulation in leaves but no ameliorating effects on shoot growth were found in the presence of Cd 〚3〛.

Upon exposure to Cd, most species initially show a decrease in GSH, probably because of an enhanced demand of GSH for Cd detoxification 〚27〛, 〚39〛. The transient loss of GSH may be a critical step in Cd toxicity because we found that the depletion in GSH was also accompanied by severe diminution of ascorbate peroxidase, catalase, and glutathione reductase activities and a significant accumulation of H2O2 〚31〛. Metabolic modelling suggested that this loss in antioxidative defences was sufficient to explain the observed H2O2 accumulation 〚25〛, 〚31〛. The formation of H2O2 after Cd exposure has been detected in different species and experimental systems such as potato tuber discs, suspension cultures of tobacco cells, and pine roots 〚24〛, 〚32〛, 〚34〛. These observations indicate that Cd, though not a transition metal, commonly causes oxidative stress in plants. Since H2O2 is a signalling molecule which triggers, among other responses, secondary pathways and programmed cell death, we suggested that Cd may affect the cellular redox control via H2O2 accumulation, thereby, inducing common plant defences and cell death 〚32〛.

Fast-growing trees species of the genus Populus are currently under discussion for soil reclamation in heavy metal-polluted sites. A prerequisite for Cd tolerance is the ability of roots to sustain growth and maintain cellular homeostasis in heavy metal-polluted soil. In the present study, we put forward the hypothesis that Cd-induced injury is related to the disturbance of cellular redox control in root tips. To address this idea, Cd-induced changes in antioxidative systems were investigated in relation to H2O2 accumulation, oxidative stress, and growth of poplar (Populus × canescens, hybrid of P. tremula × P. alba) roots. In addition, we tested whether exposure to H2O2 had effects on root antioxidant systems and growth similar to those observed after Cd exposure.

Section snippets

Exposure to Cd has negative effects on growth and causes oxidative stress

When hydroponically grown poplar plants were exposed to 5 μM Cd, cadmium accumulated to tissue concentrations of about 500 μg g–1 dry mass within 6 h but did not increase significantly thereafter (Fig. 1A). This observation suggests that the cellular metal homeostasis can be maintained in environments with low Cd concentrations. By contrast, exposure to 50 μM Cd resulted in about four-fold higher Cd concentrations after 6 h than exposure to 5 μM Cd and resulted in further significant increases

Growth and antioxidant systems in the presence of low Cd concentrations

An important result of the present study was that poplar roots in the presence of low Cd concentrations showed growth stimulation in comparison with controls (Fig. 1B). It has occasionally been reported that low, sub-lethal concentrations of heavy metals caused increased root growth 〚15〛. The reasons for this stimulation are not known. The threshold for toxicity, which leads to growth inhibition, is species-specific. For example in pine seedlings (Pinus sylvestris), grown under almost identical

Culture of plants, experimental treatments and harvest

Microcuttings of P. × canescens (a hybrid of P. tremula × P. alba) were propagated by micropropagation 〚33〛. The plantlets were grown at day/night conditions of 21 °C air temperature, 17:00–07:00 hours day length under white light of 250 μmol m–2 s–1 photosynthetic photon flux (Osram L18 W/21 lamps). After 3 weeks, the plantlets were transferred to aerated nutrient solutions 〚11〛. The solution was changed every 5 days. After 3 weeks of acclimation, the plants were treated with 5 or 50 μM CdSO4

Acknowledgments

We thank the Deutscher Akademischer Austausch dienst (DAAD) for funding a scholarship to P. Nikolova. We are grateful to S. Elend for excellent technical assistance.

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