Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2001-08-20
2003-06-10
Nelson, Amy J. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S278000, C800S288000, C800S298000, C435S069100, C435S468000, C435S419000
Reexamination Certificate
active
06576816
ABSTRACT:
INTRODUCTION
1. Field of the Invention
The field of the invention is enhanced heavy metal phytoremediation by genetically engineered plants.
2. Background of the Invention
Heavy metals and metalloids such as cadmium, lead and mercury are an increasing environmental problem worldwide. Green plants can be used to remove heavy metals by sequestrating, stabilizing or biochemically transforming them. This cost-effective and environment-friendly technology has been called phytoremediation (Salt et al., 1995, Biotechnology 13:468-474). Hyperaccumulators—heavy metal accumulating flora collected from metal-contaminated sites—offer one option for the phytoremediation of metal-contaminated sites. However, these hyperaccumulators tend to grow slowly and produce little biomass. An alternative approach is to genetically engineer fast-growing species to improve their metal tolerance and metal accumulating capacity.
Non-protein thiols (NPTs), which contain a high percentage of cysteine sulfhydryl residues in plants, play a pivotal role in heavy metal detoxification. Glutathione (&ggr;-Glu-Cys-Gly, GSH) is one of the most important components of NPT metabolism. GSH is thought to play multiple roles in heavy metal detoxification: it protects cells from oxidative stress damage; glutathione is a substrate for glutathione S-transferases and enables neutralization of heavy metals (Marrs, 1996, Annu Rev Plant Physiol Plant Mol Biol 47:127-158); and GSH is the direct precursor of phytochelatins (PCs). Phytochelatins are heavy metal-binding peptides involved in heavy metal tolerance and sequestration (Steffens, 1990). They comprise a family of peptides with the general structure (&ggr;-Glu-Cys)
n
-Gly, where n=2 to 11 (Rauser, 1995, Plant Physiol. 109: 1141-49). Phytochelatins were shown to be induced by heavy metals such as cadmium (Cd) in all plants tested (Zenk, 1996, Gene 179:21-30), including Indian mustard (Speiser et al. 1992, Plant Physiol. 99:817-821). The roles of glutathione in heavy metal tolerance and phytochelatin synthesis were illustrated in Cd-sensitive mutants of
Arabidopsis thaliana.
For example, the Cd-sensitive cad2 mutant was defective in glutathione biosynthesis (Howden et al., 1995, Plant Physiol 107:1067-1073). Glutathione is synthesized from its constituent amino acids in two sequential, ATP-dependent enzymatic reactions, catalyzed by &ggr;-glutamyl-cysteine synthetase (ECS) and glutathione synthetase (GS), respectively. Phytochelatin synthase subsequently catalyzes the elongation of the (&ggr;-Glu-Cys)
n
by transferring a &ggr;-Glu-Cys group to glutathione or to PCs (Zenk, 1996; Chen et al., 1997, Physiol Plant 101:165-172).
Overexpressing the
Escherichia coli
gshII gene, encoding glutathione synthetase, in Indian mustard gave rise to plants with increased Cd tolerance and Cd accumulation (Zhu et al. 1999, Plant Physiol 119:73-79). These Indian mustard GS plants showed increased levels of glutathione and phytochelatins relative to untransformed plants, but only in the presence of heavy metals, confirming a series of reports indicating that GS is rate limiting for glutathione synthesis only in the presence of heavy metals. For example, over-expression of the
E. coli
gshII gene, encoding GS, does not normally increase foliar glutathione levels in poplar, but under heavy metal stress the regulation of glutathione biosynthesis undergoes a significant change. Heavy metals activate the PC synthase enzyme and thus induce the biosynthesis of PCs, resulting in a depletion of cellular glutathione levels (Zenk, 1996). Consequently, the feed-back inhibition of ECS by glutathione is released. Furthermore, ECS expression may be enhanced by heavy metals. It was demonstrated that Cd enhances the transcription of the ECS gene (Hatcher et al. 1995). In contrast, Cd may deactivate GS, as GS activity has previously been shown to be inhibited by Cd while the same Cd treatment had no affect on ECS activity (Schneider and Bergmann, 1995). Exposure of maize roots to Cd, besides causing a decline in GSH, caused an accumulation of &ggr;-Glu-Cys, possibly because the activity of GSH synthetase was reduced in vivo (Rauser et al., 1991, Plant Physiol 97:128-138). Therefore, under Cd stress, the GS enzyme becomes rate-limiting for the biosynthesis of glutathione and PCs, and thus over-expression of gshII can alleviate the depletion of glutathione and enhance PC synthesis.
Though in the presence of heavy metals GS is rate limiting, in the absence of heavy metals ECS is rate-limiting (Zhu et al., 1999). For example, overexpression in poplar of the
E. coli
gshI gene, encoding g-ECS, resulted in increased foliar glutathione levels (Arisi et al., 1997, Planta 203:362-372). In the presence of heavy metals, expression of tomato ECS restored some degree of heavy metal tolerance to the cad2
Arabidopsis thaliana
mutant. However, over-expression of this gene did not increase the Cd tolerance of wild type
A. thaliana
plants (Goldsbrough, 1999, Metal tolerance in plants: the role of phytochelatins and metallothioneins. In N Terry, G S Banuelos, eds, Phytoremediation of Trace Elements. Ann Arbor Press, Ann Arbor, Mich.) and reportedly failed to increase Cd accumulation in poplars (Noctor et al., 1998, J Exp Bot 49:523-647, 640). Nevertheless, based on fortuitous discoveries in our laboratory, we sought to obtain fast-growing plants with superior heavy metal accumulation and tolerance for phytoremediation by overexpressing ECS. We have successfully developed transgenic plants that have an increased ability for heavy metal accumulation and tolerance. These ECS plants greatly enhance the efficiency of heavy metal phytoextraction from polluted soils and wastewater.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for heavy metal phytoremediation. The subject compositions include plants which are genetically engineered to overexpress glutamylcysteine synthetase and thereby provide enhanced heavy metal accumulation. In particular embodiments, the plants comprise a gene encoding ECS operably linked to a heterologous promoter, the plant is a member of the Brassicaceae family, such as
Brassica juncea,
and/or the heavy metal is cadmium. In general, the subject methods comprise the steps of growing such plants in a medium such as soil or water comprising a heavy metal, under conditions wherein the ECS is overexpressed, whereby the plant provides enhanced accumulation of the heavy metal, whereby the heavy metal content of the medium is decreased.
REFERENCES:
patent: 5364451 (1994-11-01), Raskin et al.
patent: 5747329 (1998-05-01), Meister et al.
FEBS Letter (1998), 427(1):129-133.*
EMBO Journal (1997), 16(11):3057-3065.*
Journal of Biological Chemistry (1996), 271(29):17485-17490.*
PNAS (1998), 95(12049:12054).*
FEBS Letters (1997), 404:216-220.*
Watanabe et al. Nucleic Acids Research, Nov. 1986 14:4393-40.
Arisi et al. Planta, 1997 203:362-72.
Chen et al. Plant Physiol. 1994 106:233-39.
Noctor et al. Journal of Experimental Botany, Apr. 1998 49(321):623-47.
Peter Goldsbrough, Metal Tolerance in Plants: The role of phytochelatins and Metallothioneins. Ann Arbor Press, 1999 221-28.
Zhu et al. Plant Physiol. 1999 119:73-79.
Pilon-Smits Elizabeth
Terry Norman
Zhu Yong Liang
Ibrahim Medina A.
Nelson Amy J.
Osman Richard Aron
The Regents of the University of California
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