Arazi, T; Sunkar, R; Kaplan, B; Fromm, H*

Plant Journal [Plant J.], vol. 20, no. 2, pp. 171-182, Oct 1999

All organisms require a minimal amount of metal ions to sustain their metabolism, growth and development. At the same time, their intrinsic metal-uptake systems render them vulnerable to toxic levels of metals in the biosphere. Using radiolabeled recombinant calmodulin as a probe to screen a tobacco cDNA library, we have discovered a protein designated NtCBP4 (Nicotiana tabacum calmodulin-binding protein) that can modulate plant tolerance to heavy metals. Structurally, NtCBP4 is similar to vertebrate and invertebrate K super(+) and to non-selective cation channels, as well as to recently reported proteins from barley and Arabidopsis. Here we report on the subcellular localization of NtCBP4 and the phenotype of transgenic plants overexpressing this protein. The localization of NtCBP4 in the plasma membrane was manifested by fractionating tobacco membranes on sucrose gradients or by aqueous two-phase partitioning, and subsequently using immunodetection. Several independent transgenic lines expressing NtCBP4 had higher than normal levels of NtCBP4. These transgenic lines were indistinguishable from wild type under normal growth conditions. However, they exhibited improved tolerance to Ni super(2+) and hypersensitivity to Pb super(2+), which are associated with reduced Ni super(2+) accumulation and enhanced Pb super(2+) accumulation, respectively. To our knowledge this is the first report of a plant protein that modulates plant tolerance or accumulation of Pb super(2+). We propose that NtCBP4 is involved in metal uptake across the plant plasma membrane. This gene may prove useful for implementing selective ion tolerance in crops and improving phytoremediation strategies.

Bizily, SP; Rugh, CL; Summers, AO; Meagher, RB

Proceedings of the National Academy of Sciences, USA [Proc. Natl. Acad. Sci. USA], vol. 96, no. 12, pp. 6808-6813, 8 Jun 1999

Methylmercury is an environmental toxicant that biomagnifies and causes severe neurological degeneration in animals. It is produced by bacteria in soils and sediments that have been contaminated with mercury. To explore the potential of plants to extract and detoxify this chemical, we engineered a model plant, Arabidopsis thaliana, to express a modified bacterial gene merBpe, encoding organomercurial lyase (MerB) under control of a plant promoter. MerB catalyzes the protonolysis of the carbon---mercury bond, removing the organic ligand and releasing Hg(II), a less mobile mercury species. Transgenic plants expressing merBpe grew vigorously on a wide range of concentrations of monomethylmercuric chloride and phenylmercuric acetate. Plants lacking the merBpe gene were severely inhibited or died at the same organomercurial concentrations. Six independently isolated transgenic lines produced merBpe mRNA and MerB protein at levels that varied over a 10- to 15-fold range, and even the lowest levels of merBpe expression conferred resistance to organomercurials. Our work suggests that native macrophytes (e.g. trees, shrubs, grasses) engineered to express merBpe may be used to degrade methylmercury at polluted sites and sequester Hg(II) for later removal.

Zhu, YL; Pilon-Smits, EAH; Jouanin, L; Terry, N

Plant Physiology [Plant Physiol.], vol. 119, no. 1, pp. 73-80, Jan 1999

An important pathway by which plants detoxify heavy metals is through sequestration with heavy-metal-binding peptides called phytochelatins or their precursor, glutathione. To identify limiting factors for heavy-metal accumulation and tolerance, and to develop transgenic plants with an increased capacity to accumulate and/or tolerate heavy metals, the Escherichia coli gshII gene encoding glutathione synthetase (GS) was overexpressed in the cytosol of Indian mustard (Brassica juncea). The transgenic GS plants accumulated significantly more Cd than the wild type: shoot Cd concentrations were up to 25% higher and total Cd accumulation per shoot was up to 3-fold higher. Moreover, the GS plants showed enhanced tolerance to Cd at both the seedling and mature- plant stages. Cd accumulation and tolerance were correlated with the gshII expression level. Cd-treated GS plants had higher concentrations of glutathione, phytochelatin, thiol, S, and Ca than wild-type plants. We conclude that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and phytochelatins, and that overexpression of GS offers a promising strategy for the production of plants with superior heavy-metal phytoremediation capacity.

Salt, DE

Nature Biotechnology [Nat. Biotechnol.], vol. 16, no. 10, p.905, Oct 1998

In a twist of fate, probably never envisioned by our forbears, we may soon be using trees to help us "heal the hurt" inflicted on the earth. By borrowing genes from bacteria, Rugh et al. have been able to make yellow poplar perform a trick alchemists learned long ago: Turning cinnabar into quicksilver - or, in modern chemical parlance, reducing ionic mercury to volatile, elemental mercury. This trick raises the possibility that trees may soon be able to remove poisonous mercury from soil by blowing it into the atmosphere, in a process aptly named phytovolatilization (phyto, plant; volatilization, change to a vapor).

Rugh, CL; Senecoff, JF; Meagher, RB; Merkle, SA*

Nature Biotechnology [Nat. Biotechnol.], vol. 16, no. 10, pp. 925-928, Oct 1998

We examined the ability of yellow poplar (Liriodendron tulipifera) tissue cultures and plantlets to express modified mercuric reductase (merA) gene constructs. Mercury-resistant bacteria express merA to convert highly toxic, ionic mercury, Hg(II), to much less toxic, elemental mercury, Hg(0). Expression of merA in transgenic plants might provide an ecologically compatible approach for the remediation of mercury pollution. Because the alteration of the bacterial merA gene sequence is necessary for high-level expression in Arabidopsis thaliana, yellow poplar proembryogenic masses (PEMs) were transformed with three modified merA constructs via microprojectile bombardment. Each construct was synthesized to have altered flanking regions with increasing amounts of modified coding sequence. All merA constructs conferred resistance to toxic, ionic mercury in independently transformed PEM colonies. Stability of merA transgene expression increased in parallel with the extent of gene coding sequence modification. Regenerated plantlets containing the most modified merA gene (merA18) germinated and grew vigorously in media containing normally toxic levels of ionic mercury. The merA18 plantlets released elemental mercury at approximately 10 times the rate of untransformed plantlets. These results indicate that plants expressing modified merA constructs may provide a means for the phytoremediation of mercury pollution.

Heaton, ACP; Rugh, CL; Wang, Nian-jie; Meagher, RB

Journal of Soil Contamination [J. Soil Contam.], vol. 7, no. 4, pp. 497-509, Jul 1998

Inorganic mercury in contaminated soils and sediments is relatively immobile, though biological and chemical processes can transform it to more toxic and bioavailable methylmercury. Methylmercury is neurotoxic to vertebrates and is biomagnified in animal tissues as it is passed from prey to predator. Traditional remediation strategies for mercury contaminated soils are expensive and site-destructive. As an alternative we propose the use of transgenic aquatic, salt marsh, and upland plants to remove available inorganic mercury and methylmercury from contaminated soils and sediments. Plants engineered with a modified bacterial mercuric reductase gene, merA, are capable of converting Hg(II) taken up by roots to the much less toxic Hg(0), which is volatilized from the plant. Plants engineered to express the bacterial organomercurial lyase gene, merB, are capable of converting methylmercury taken up by plant roots into sulfhydryl-bound Hg(II). Plants expressing both genes are capable of converting ionic mercury and methylmercury to volatile Hg(0) which is released into an enormous global atmospheric Hg(0) pool. To assess the phytoremediation capability of plants containing the merA gene, a variety of assays were carried out with the model plants Arabidopsis thailana, and tobacco (Nicotiana tabacum).

Chaney, RL; Malik, M; Li, Yin M; Brown, SL; Brewer, EP; Angle, JS; Baker, AJM

Current Opinion in Biotechnology [CURR. OPIN. BIOTECHNOL.], vol. 8, no. 3, pp. 279-284, Jun 1997

The phytoremediation of metal-contaminated soils offers a low-cost method for soil remediation and some extracted metals may be recycled for value. Both the phytoextraction of metals and the phytovolatilization of Se or Hg by plants offer great promise for commercial development. Natural metal hyperaccumulator phenotype is much more important than high-yield ability when using plants to remove metals from contaminated soils. The hypertolerance of metals is the key plant characteristic required for hyperaccumulation; vacuolar compartmentalization appears to be the source of hypertolerance of natural hyperaccumulator plants. Alternatively, soil Pb and Cr super(5+) may be inactivated in the soil by plants and soil amendments (phytostabilization). Little molecular understanding of plant activities critical to phytoremediation has been achieved, but recent progress in characterizing Fe, Cd and Zn uptake by Arabidopsis and yeast mutants indicates strategies for developing transgenic improved phytoremediation cultivars for commercial use.

Rugh, CL; Wilde, HD; Stack, NM; Thompson, DM; Summers, AO; Meagher, RB*

Proceedings of the National Academy of Sciences, USA [PROC. NATL. ACAD. SCI. USA], vol. 93, no. 8, pp. 3182-3187, 1996

With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg super(2+) to the less toxic, relatively inert metallic mercury (Hg super(0)). The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl sub(2) concentrations of 25-100 mu M (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg super(0) relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au super(3+). These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution.