Phytoremediation of arsenate contaminated soil by transgenic canola and the plant growth-promoting bacterium Enterobacter cloacae CAL2

doi:10.1016/S0981-9428(02)01375-X

Plant Physiology and Biochemistry

Volume 40, Issue 4, April 2002, Pages 355-361

https://doi.org/10.1016/S0981-9428(02)01375-X Get rights and content

Abstract

It was previously observed that transgenic tomato plants that express the Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylate (ACC) deaminase (EC 4.1.99.4) gene, and thereby produce lower levels of ethylene, were partially protected from the deleterious effects of six different metals. However, since tomato plants are unlikely to be utilized in the phytoremediation of contaminated terrestrial sites, transgenic canola (Brassica napus) plants that constitutively express the same gene were generated and tested for their ability to proliferate in the presence of high levels of arsenate in the soil and to accumulate it in plant tissues. The ability of the plant growth-promoting bacterium E. cloacae CAL2 to facilitate the growth of both non-transformed and ACC deaminase-expressing canola plants was also tested. In the presence of arsenate, in both the presence and absence of the added plant growth-promoting bacterium, transgenic canola plants grew to a significantly greater extent than non-transformed canola plants.

Introduction

Phytoremediation is defined as the use of green plants to remove pollutants from the environment, or to render them harmless 〚8〛, 〚33〛. Phytoremediation includes: phytoextraction, the accumulation of organic compounds or metals from the soil in plant tissue 〚24〛; rhizofiltration, whereby plant roots absorb, concentrate or precipitate pollutants from effluents; phytostabilization, the reduction in the mobility of pollutants by absorption and precipitation by plants, thus reducing their bioavailability; and phytovolatilization, the use of plants to volatilize pollutants 〚34〛.

Metal-hyperaccumulating plants can tolerate and concentrate high levels of heavy metals. However, the development of these plants for phytoextraction has been limited by the relative small size and slow growth rate of the plants. The ideal plant for phytoextraction should grow rapidly, produce a high amount of biomass, and be able to tolerate and accumulate high concentrations of metals in shoots. The efficiency of phytoextraction is also influenced by the bioavailability of metals to plants in soil. In some cases, this problem can be solved by applying chemical chelating agents to the soil in order to increase the metal solubility 〚3〛. Enhanced rates of metal ion translocation from roots to shoots are also important for efficient phytoextraction. Shoots provide more space for metal storage and are easily harvested. Increased plant transpiration may promote metal mobility from roots to shoots. Thus, chemically mutagenized Brassica juncea plants with increased levels of leaf transpiration accumulated twice as much lead as the wild-type 〚13〛.

Arsenic is one of the metal contaminants of soil that requires remediation. Arsenic affects seed germination, and reduces root length and mass 〚12〛. Arsenite is 4–100 times more toxic than arsenate. The amounts of arsenic found in plant tissue are generally proportional to its level in soil 〚30〛.

Some plant growth-promoting bacteria can significantly increase the growth of plants in the presence of heavy metals including nickel, lead and zinc 〚5〛, 〚6〛 by lowering the level of stress-induced ethylene, thus allowing plants to develop longer roots and better establish during early stages of growth 〚17〛. Once the seedling is established, the bacterium can also help the plant to acquire sufficient iron for optimal plant growth 〚6〛. In other experiments, transgenic tomato plants that express Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylate (ACC) deaminase (EC 4.1.99.4), and thereby produce lower levels of ethylene, were shown to be partially protected from the deleterious effects of six different metals 〚18〛.

In this study, transgenic canola (Brassica napus cv. Westar) plants that express E. cloacae UW4 ACC deaminase were constructed and tested for the ability to proliferate in the presence of arsenate. The ability of the plant growth-promoting bacterium, E. cloacae CAL2, to facilitate the growth of both non-transformed and ACC deaminase-expressing canola plants was also tested.

Section snippets

Results and discussion

Genomic DNA from transgenic canola (Brassica napus cv. Westar) was digested with HindIII and Southern analysis was performed to determine the number of sites at which the ACC deaminase gene had integrated into the plant genome. Since HindIII does not cut within the ACC deaminase gene, the number of bands on a Southern blot corresponds directly to the number of integrated ACC deaminase genes, and the single band observed with transformant C1-1 (Fig. 1) is consistent with integration of a single

Discussion

Transgenic canola plants expressing a bacterial ACC deaminase were used as part of a system to deal with arsenate in the soil. This approach is based on previous observations that transgenic tomato plants expressing a bacterial ACC deaminase gene lower stress-induced ethylene levels 〚1〛, 〚22〛. As a consequence, these plants can tolerate a number of different environmental stresses including heavy metals 〚18〛, fungal phytopathogens 〚26〛 and flooding 〚20〛.

Regardless of the presence or absence of

Bacterium and media

Enterobacter cloacae CAL2 was isolated from a rhizosphere soil sample based on its ability to utilize ACC as a sole nitrogen source 〚15〛. This ACC deaminase-containing bacterium promotes canola root elongation, and produces IAA, siderophores and antibiotics 〚35〛, 〚40〛. The bacterium was grown at 30 °C on Tryptic Soybean Broth (TSB) medium (Difco; Detroit, MI) or on DF salts minimal medium 〚11〛 with either 0.2% (NH₄)₂SO₄ (w/v) or 3 mM ACC (Calbiochem, La Jolla, CA) as a nitrogen source.

Plasmid construction

The ACC

Acknowledgements

We are grateful to CRESTech (a Province of Ontario Centre of Excellence), Ontario Hydro (Toronto, Ontario, Canada) and the Natural Sciences and Engineering Research Council of Canada for providing funds in support of this research. Thanks to Dr. Kimberly Kenward and Dr. Donna Penrose for critically reviewing the manuscript, and to Corrine Collard for technical assistance.

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¹: These two authors contributed equally to the work described in this manuscript.

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Phytoremediation of arsenate contaminated soil by transgenic canola and the plant growth-promoting bacterium Enterobacter cloacae CAL2

Abstract

Introduction

Section snippets

Results and discussion

Discussion

Bacterium and media

Plasmid construction

Acknowledgements

J. Theor. Biol.

J. Biotechnol.

Plant Physiol. Biochem.

Curr. Opin. Biotechnol.

Curr. Opin. Biotechnol.

Dormancy and the control of germination

Enhanced accumulation of Pb in Indian mustard by soil-applied chelating agents

Environ. Sci. Technol.

A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding

Anal. Biochem.

A plant growth-promoting bacterium that decreases nickel toxicity in seedlings

Appl. Environ. Microbiol.

Plant growth-promoting bacteria that decrease heavy metal toxicity in plants

Can. J. Microbiol.

Design and construction of a versatile system for the expression of foreign genes in plants

Gene

Remediation of contaminated soils with green plants: an overview

In Vitro Cell. Dev. Biol.

A plant DNA mini-preparation: version II

Plant Mol. Biol. Rep.

Broad host range DNA cloning system for Gram negative construction of a gene bank of Rhizobium meliloti

Proc. Natl. Acad. Sci. USA

Experiments with some microorganisms which utilize ethane and hydrogen

J. Bacteriol.

Effect of Pb, Cd, Hg, As, and Cr on germination and root growth of Sinapis alba seeds

Bull. Environ. Contam. Toxicol.

Use of plant roots for phytoremediation and molecular farming

Proc. Natl. Acad. Sci. USA

The enhancement of plant growth by free-living bacteria

Can. J. Microbiol.

A novel procedure for rapid isolation of plant growth promoting pseudomonads

Can. J. Microbiol.

Biochemical and Genetic Mechanisms Used by Plant Growth-promoting Bacteria