Elsevier

Biochemical Engineering Journal

Volume 71, 15 February 2013, Pages 19-24
Biochemical Engineering Journal

Bacterial heavy metal transporter MerC increases mercury accumulation in Arabidopsis thaliana

https://doi.org/10.1016/j.bej.2012.11.007Get rights and content

Abstract

This study evaluated the feasibility of transgenic Arabidopsis engineered to express the bacterial heavy metal transporter MerC for the phytoremediation of mercury pollution. MerC, MerC–SYP121, or MerC–AtVAM3 proteins were found to be expressed in leaf segments of transgenic plants using an anti-MerC antibody immunostaining method. By sucrose density gradient centrifugation and immunoblotting analyses, MerC, MerC–SYP121, and MerC–AtVAM3 were found to localized in the Golgi apparatus, plasma membrane, and vacuole membrane, respectively. Transgenic Arabidopsis plants that expressed merC–SYP121 were more resistant to mercury and accumulated significantly more of this metal than wild-type Arabidopsis. These results demonstrated that expression of the bacterial heavy metal transporter MerC promoted the transport and accumulation of mercury in transgenic Arabidopsis, which may be a useful method for improving plants for the phytoremediation of mercury pollution.

Highlights

► This study showed that transgenic MerC-SNARE plants may be useful for mercury phytoremediation. ► MerC is a bacterial heavy metal transporter and SNARE is used as an organelle-targeting marker to direct MerC to specific membranes. ► Transgenic plants were more resistant to mercury and accumulated more mercury. ► MerC can promote the transport and accumulation of mercury in transgenic plants.

Introduction

Mercury is a toxic non-essential metal that causes severe physiological effects when ingested at relatively small doses. The primary mechanism of cellular toxicity is the reaction of mercury with reduced sulfhydryl residues on critical proteins. The binding of mercury to these macromolecules inhibits their normal biological functions and disrupts cellular metabolism [1]. Histological studies have confirmed that mercury poisoning can lead to atrophy of the central nervous system [2]. The serious adverse effects of this contaminant mean that there is an urgent need to develop an effective and affordable technology for its removal from the environment.

Phytoremediation refers to the use of green plants in the removal of environmental pollutants, e.g., 2–10 ppm Cd [3], [4], and it is recognized as a cost effective, sustainable, and environmentally friendly approach that offers great advantages during the large scale clean-up of contaminated sites [5], [6]. Recent studies of addressing heavy metal removal from contaminated soils has focused on the development of genetically engineered plants with an increased ability to transport and accumulation of specific metals, which are mediated by a metal transporter, but without succumbing to the metal toxicity themselves. Previously, we reported the eukaryotic expression of a bacterial heavy metal transporter, i.e., MerC in the Tn21-encoded mer operon, which can recognize and transport cadmium and mercury into bacterial cells. MerC was expressed in a model eukaryotic organism, i.e., yeast, which resulted in increased mercury accumulation from a mercury-contaminated medium [7]. We also demonstrated that Arabidopsis soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs) [8], [9], [10] can be used as organelle-targeting markers to direct the bacterial heavy metal transporter MerC to specific membranes in yeast [7], [11] and Arabidopsis suspension-cultured cells [12]. Among Arabidopsis thaliana SNARE molecules, SYP121 is involved in the transport of secretory vesicles at the plasma membrane, and AtVAM3 (SYP22) provides target SNARE function during the late stages of vacuolar assembly [13]. Recently, we found that transgenic yeast [11] and Arabidopsis [12] expressing MerC in the plasma membrane accumulated significantly more cadmium than their respective wild types. Thus, SNARE-mediated targeting of MerC to the plasma membrane may represent a promising strategy for improving the efficiency and potential biomass accumulation during the remediation of cadmium pollution.

The current study evaluated the feasibility of transgenic Arabidopsis plants engineered to express MerC-SNAREs for use in the phytoremediation of mercury pollution.

Section snippets

Materials and growth conditions

Arabidopsis thaliana ecotype Columbia (wild type), the T3–5 progeny of merC, merC–SYP121, and merC–AtVAM3 transgenic plants, was engineered to express a bacterial heavy metal transporter, MerC, and plant SNARE [12]. Plants were grown on Jiffy-7 peat pellets while surface-sterilized seeds were sown onto MS (Murashige and Skoog medium) agar or MS gellan gum plates. All plants were grown in an environmental growth chamber (Sanyo, Tokyo, Japan) at 22 °C under long-day (16:8 h light:dark) conditions.

Enzymes and reagents

Results

In a previous report [12], we tested the potential of plants for accumulating heavy metals such as cadmium from contaminated sites by engineering an Arabidopsis plant that expressed a bacterial heavy metal transporter MerC fused with SNARE, SYP121, or AtVAM3. Seventeen merC, 20 merC–SYP121 and 15 merC–AtVAM3 independent transgenic lines were obtained by selection on MS medium containing 50 mg L−1 kanamycin.

Discussion

A strategic focus for improving the bioremediation potential of biomass, such as bacteria, fungi, and plants, is the introduction of genes that promote the uptake, transport and sequestration of pollutants. In a previous study, we reported that yeast SNAREs, Sso1p, and Vam3p, and plant SNAREs, SYP111, and AtVAM3 (SYP22), could serve as useful molecular tools for targeting the bacterial heavy metal transporter MerC to the plasma and vacuolar membranes of yeast [7], [11]. In addition, we found

Summary and conclusions

The present study evaluated the feasibility of transgenic Arabidopsis engineered to express the bacterial heavy metal transporter MerC for the phytoremediation of mercury pollution. The results suggest that the expression of the MerC can promote the transport and accumulation of mercury in transgenic plants, which may be a useful method for improving plants for the phytoremediation of mercury pollution.

Acknowledgments

We are grateful to Dr. M. Sugiyama of the University of Tokyo for the gift of Arabidopsis thaliana Columbia. We thank Dr. S. Uraguchi of the University of Tokyo for valuable advice. We also thank Miss A. Iida, Miss C. Hirota, Miss M. Kuhara, and Miss M. Kaburagi for technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research (C) (No. 21510088) to M.K. from the Ministry of Education, Science and Culture, Japan. This work was also supported in part by a

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