Research article
Cadmium tolerance in Brassica juncea roots and shoots is affected by antioxidant status and phytochelatin biosynthesis

https://doi.org/10.1016/j.plaphy.2012.05.002Get rights and content

Abstract

Indian mustard (Brassica juncea L. Czern.) tolerates high concentrations of heavy metals and is a promising species for the purpose of phytoextraction of cadmium (Cd) from metal-contaminated soils. This work investigates the extent to which antioxidant and metal sequestering mechanisms are responsible for this tolerance. To this end, seedlings of Indian mustard were grown for 7 days in 0, 50 or 200 μM Cd. Increasing Cd concentrations led to a progressive Cd accumulation in roots and shoots, accompanied by an organ-dependent alteration in mineral uptake, and a decrease in root/shoot length and fresh/dry weight. Cd negatively affected chlorophyll and carotenoid contents and activated the xanthophyll cycle, suggesting the need to protect the photosynthetic apparatus from photoinhibition. Shoots seemed to be less efficient than roots in ROS scavenging, as indicated by the different response to Cd stress shown by peroxidase and catalase activities and, solely with regard to the highest Cd concentration, by ascorbate level. Such a different antioxidant capacity might at least partly explain differences in the trend of lipid peroxidation observed in the two organs. Moreover, in both roots and shoots, glutathione and phytochelatin content markedly increased under Cd stress, regardless of the metal concentration involved.

Highlights

► Seedlings of Indian mustard were grown for 7 days in 0, 50 or 200 μM Cd(NO3)2. ► Antioxidant and metal sequestering mechanisms were studied in roots and shoots. ► Cadmium decreased chlorophyll and carotenoid content and activated xanthophyll cycle. ► Shoots were less efficient to enhance antioxidant defenses than roots. ► In both organs glutathione and phytochelatin content increased under Cd stress.

Introduction

Although not essential for plants, in several species cadmium (Cd) is easily taken up by roots, with translocation and accumulation in the shoots occurring readily [1]. In planta, Cd can cause various symptoms of phytotoxicity such as browning of root tips, inhibition of root elongation, reduction of biomass, chlorosis and even death, owing to its influence on different metabolic processes such as water and nutrient uptake, photosynthesis and respiration [2]. Cd toxicity has been attributed to several factors, i.e. the blockage of essential functional groups in biomolecules [3]; displacement of essential metal ions from enzymes [4]; enhanced accumulation of reactive oxygen species (ROS) such as superoxide radical (O2·-), hydroxyl radical (OH·) and hydrogen peroxide (H2O2), because of ROS-increased production and/or reduced detoxification [5], [6], [7].

To limit and circumscribe Cd toxicity, plants provide several cellular defense mechanisms, such as plasma membrane exclusion, vacuolar compartmentalization and cell wall immobilization. Thiol-peptide compounds, which on average contain an elevated percentage of cysteine (Cys) sulfhydryl residues, play a pivotal role in heavy metal detoxification. In fact, the reduced form of glutathione (γ-Glu-Cys-Gly, GSH) is essential in metal tolerance and sequestration, by protecting cells from oxidative stress damage [8] and by binding several metals [9]. GSH is also the direct precursor of phytochelatins (PCs), the principal heavy metal-complexing peptides of plants [10]. PCs comprise a family of peptides with the general structure (γ-Glu-Cys)n-Gly, where n = number of repetitions of the γ-Glu-Cys unit, which can vary from 2 to 11 (more commonly from 2 to 5) [10]. PCs reduce the free metal concentration in the cytosol by binding and transporting the metal to specific compartments, mainly the vacuole, and possess a high antioxidant capacity [11]. PCs synthesis has been shown to be induced by Cd in all plants tested, including Indian mustard [12].

Plants actually possess several protective mechanisms for coping with ROS, involving enzymes such as superoxide dismutases (SOD), peroxidases (POD) and catalases (CAT), as well as antioxidant metabolites such as ascorbate, glutathione and α-tocopherol [8]. A pivotal defensive role against metal-induced oxidative stress is played by the ascorbate–glutathione cycle, which reduces H2O2 at the expense of ascorbate in an ascorbate peroxidase (APX)-dependent reaction, and recycles ascorbate in the reduced form by dehydroascorbate reductase using GSH as a substrate. Oxidized glutathione is, in turn, reduced by glutathione reductase in the presence of NAD(P)H [8].

Indian mustard (Brassica juncea, Brassicaceae) can store high concentrations of heavy metals in its organs [13], [14]. This species might thus be involved in phytoremediation, either directly as a phytoremediation crop, or indirectly, as a source of genes for improvement of other phytoremediation-targeted plants. In general, there are many plant species that have evolved means of adapting to extreme soil metal environments [13]. In actual fact, about 450 species from a number of different families such as the Brassicaceae, Caryophyllaceae, and Fabaceae possess the ability to tolerate very high levels of metal(loid)s in the soil, and, more importantly, in shoots [15]. The Brassicaceae is the best represented family among these metal-accumulator families, and B. juncea (although not being enumerated amongst Cd hyperaccumulators sensu stricto, as it does not naturally grow in metalliferous sites [16]), might represent a strong tool for developing effective Cd phytoremediation strategies, possibly also with regard to lead and mercury soil decontamination.

In this study, we mechanistically verified whether B. juncea's considerable ability to cope with Cd stress was linked to the capacity of this species to sequester the metal by means of an elevated synthesis of thiol-peptide compounds, as well as to variations in the levels of leaf pigments, antioxidant metabolites and activities of antioxidant enzymes. In addition, potential imbalances in mineral status and lipid peroxidation levels caused by Cd were evaluated.

Section snippets

Effects of Cd on plant growth

The addition of Cd to the nutrient solution resulted in a visually marked reduction in the growth of Indian mustard plants, this being particularly evident at the highest Cd concentration (Fig. 1A). At the end of the growth period, the Cd-treated seedlings displayed leaf chlorosis. Following the addition of 50 or 200 μM Cd, shoot length decreased by around −27% and −45%, while root length decreased by approximately −17% and −31%, respectively (Fig. 1B).

Shoot and root fresh weight (mg/seedling)

Discussion

In this study, we investigated growth and metabolite profiling in roots and shoots of Indian mustard plants exposed to 50 and 200 μM Cd for 7 days. Our growth data are consistent with the results of Guo and Marschner [17], who reported that the inhibition of root elongation of different plant species is one of the most sensitive parameters of Cd toxicity.

The retention or immobilization of high amounts of Cd in the roots is a typical response of several plants, and this mechanism can be regarded

Design, growth conditions and Cd treatments

Seeds of Indian mustard (B. juncea L. Czern.) were kindly provided by the Horticulture Crop Department - Agriculture Research Center, Ministry of Agriculture, Cairo, Egypt. The experiments were carried out in a growth chamber (16 h photoperiod, photosynthetic photon flux density of 350 μmol photons m−2 s−1, supplied by fluorescent lamps, day/night temperatures of 25/20 °C, and relative humidity of 70%). Seeds were germinated on filter paper moistened with deionized water. Following germination,

Acknowledgments

This work was supported by the Executive Programme of Scientific and Technological Cooperation between Italy-Egypt for the years 2008–2010 “Phytoremediation of heavy metal-contaminated soils by Mediterranean hyperaccumulating plants” and by funds from the Universities of Pisa and Parma, Italy.

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