Original article
Higher levels of ectopic expression of Arabidopsis phytochelatin synthase do not lead to increased cadmium tolerance and accumulation

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Abstract

Phytochelatins (PCs) are enzymatically synthesized peptides that play an important role in heavy metal homeostasis and detoxification. Recently, genes encoding PC synthase have been cloned from Arabidopsis and other species. Herein, we report on manipulating the expression of Arabidopsis thaliana PC synthase (AtPCS1) in order to increase tolerance and accumulation of cadmium in plants. Approximately 50 transgenic Arabidopsis lines have been generated following transformation with a construct containing the AtPCS1 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter (35::AtPCS1). Following screening, two lines with the highest level of expression of the 35S::AtPCS1 transgene have been selected. When seedlings of these two lines, exhibiting ~14-fold increase in level of the AtPCS1 transcript, are subjected to 85 μM CdCl2 for a 3-d period, ~30% increase in the level of PC production is observed compared to that of wild-type. However, these two transgenic lines are no more cadmium-tolerant, nor do they accumulate more cadmium than wild-type seedlings. In fact, these transgenic lines show higher sensitivity to cadmium than wild-type seedlings when the agar medium is supplemented with various concentrations of essential metals (EMs) in the presence of 75 μM CdCl2. When all remaining transgenic lines are evaluated for cadmium tolerance, regardless of their levels of AtPCS1 transgene expression, six transgenic lines exhibit approximately twofold increase in cadmium tolerance compared to wild-type plants. Surprisingly, these six cadmium-tolerant lines show only slight increases (30–40%) in expression of AtPCS1 and ~15% increase in PC content when compared to wild-type. They also show increased accumulation of cadmium in their shoots. Therefore, critical levels of AtPCS1 are important in heavy metal detoxification.

Introduction

Heavy metals have densities higher than 5 g cm–3, and include cadmium (Cd), mercury (Hg), lead (Pb), copper (Cu), and zinc (Zn), among others. These are among the most important pollutants causing worldwide environmental contamination and human health problems. Heavy metal pollutants have been discharged into the environment over the past century through various means, including mining, metal-work industries, urban traffic, power stations, and agricultural fertilizers [23].

Phytoremediation is a technology whereby green plants are used to either remove pollutants from the environment or reduce their toxicity. This technology has made rapid progress in the clean-up of heavy metal-polluted areas in a cost-effective and environmentally friendly manner [25], [28], [29]. One promising approach for phytoremediation is the utilization of naturally selected “hyperaccumulators”. These plant species accumulate extremely high levels of heavy metals in their biomass. However, most known hyperaccumulators typically accumulate only a specific element, grow slowly, and produce relatively small amounts of biomass [10]. Due to these disadvantages, genetic and molecular studies of plant defense mechanisms for heavy metal stress have been initiated to optimize and improve phytoremediation.

Plants have a number of defense mechanisms for dealing with heavy metals stress. One mechanism involves the production of cysteine-rich peptides, such as phytochelatins (PCs) and metallothioneins (MTs), for detoxification or homeostasis of heavy metals [7], [8], [27]. PCs consist of a family of enzymatically synthesized peptides having a general structure of (γ-Glu–Cys)n–Gly, where n equals 2–11 [26]. These peptides are rapidly synthesized in plants in response to exposure to toxic levels of heavy metals [6], [34]. The role of PC in heavy metal tolerance has been well characterized in Cd-sensitive mutants of Arabidopsis, cad1 and cad2 [9], [17], [18]. These mutants are deficient in PC production due to a mutation in either γ-glutamylcysteine synthetase (cad2) or in PC synthase (cad1). PCs form stable complexes with heavy metals in the cytosol, and these metal–PC complexes are subsequently sequestered into the vacuole [7], [8], [14], [30], [34]. PC synthase catalyzes the synthesis of PCs by transferring the γ-Glu–Cys moiety of glutathione (GSH) either to another GSH molecule or to a growing PC [13], [34]. Recently, genes encoding PC synthase have been cloned from Arabidopsis thaliana (AtPCS1), wheat (TaPCS1), Schizosaccharomyces pombe (SpPCS1), Caenorhabditis elegans (CePCS1), and other species [3], [4], [16], [31], [32]. The identification of PC synthase genes facilitates molecular and biochemical studies including mechanisms of enzyme activation [24], [33], tissue-specific expression [19], and transcriptional regulation [3], [21].

There are several reports on transgenic plants showing higher accumulation and tolerance for Cd. These transgenic plants carry transgenes encoding enzymes involved in cysteine, GSH or PC metabolism, such as O-acetylserine(thio)lyase [11], γ-glutamylcysteine synthetase [36], and GSH synthetase [35]. In response to Cd, these transgenic plants have been reported to have increased synthesis of either PCs or GSH. Therefore, overexpressing the PC synthase gene in transgenic plants may be a promising approach for developing plants that can be used in phytoremediation technology.

In this study, we overexpressed AtPCS1 in transgenic Arabidopsis with the goal of increasing PC synthesis, metal accumulation and metal tolerance in these plants. However, transgenic plants with a relatively high level of expression of the 35S::AtPCS1 transgene did not result in higher Cd tolerance, but rather showed higher sensitivity to Cd under some conditions. In contrast, transgenic plants showing a relatively lower level of expression of the 35S::AtPCS1 transgene demonstrated increased accumulation and tolerance of Cd compared to wild-type plants.

Section snippets

Selection of Cd-tolerant transgenic Arabidopsis expressing PC synthase (AtPCS1)

Approximately 50 independent transgenic Arabidopsis lines, designated as PCS(+) lines, expressing the C-terminal FLAG (DYKDDDL)-tagged full-length AtPCS1 cDNA (Fig. 1A) have been generated. Following screening of these lines, two T2 lines with the highest expression of the 35S::AtPCS1 transgene were identified, and evaluated for Cd tolerance. However, the progeny of these two lines did not show any enhanced tolerance to Cd when compared with wild-type seedlings. Therefore, all other transgenic

Discussion

PC plays an important role in heavy metal detoxification, and PC is synthesized from GSH by PC synthase. Efforts have been made to increase PC synthesis via manipulation of several genes involved in the PC synthesis pathway, and these were successful to producing transgenic plants that showed increased tolerance and accumulation of Cd [11], [35], [36].

In this study, we have manipulated the Arabidopsis PC synthase gene (AtPCS1), the last step in the PC synthesis pathway, for the purpose of

Plant materials, growth conditions, and treatments

Seeds of A. thaliana cv. Columbia (wild-type and transgenic PCS(+) lines) were germinated on an agar medium containing half-strength MS [22] salts and 2% (w/v) sucrose (pH 5.8) in 100 × 100 × 15 mm square plates. Plates were supplemented with EMs and CdCl2. The full-strength EM treatment (1.0×) included 25 μg l–1 CoCl2·6H2O, 17 μg l–1 CuCl2·2H2O, 19.88 mg l–1 FeCl2·4H2O, 37.26 mg l–1 EDTA, 19.79 mg l–1 MnCl2·4H2O, 0.25 mg l–1 Na2MoO4·2H2O, and 4.08 mg l–1 ZnCl2. The following EM treatments were

Acknowledgments

We thank Dr. Christopher S. Cobbett (University of Melbourne, Australia) for his generous gift of Arabidopsis mutant seeds, cad1-3, and the Arabidopsis Biological Resource Center (Columbus, Ohio) for providing us with the EST clone. This work was funded by a grant received from the Illinois Department of Natural Resources.

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