Phytochelatin synthases and uses therefor

Details Phytochelatin synthases and uses therefor Phytochelatin synthases and uses therefor

1999-07-15

2002-12-03

Fox, David T. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S298000, C800S295000, C800S288000, C800S320300, C800S306000, C435S069100, C435S320100, C435S419000, C435S468000, C536S023100, C536S023200, C536S023600, C536S023700

Reexamination Certificate

active

06489537

ABSTRACT:

BACKGROUND OF THE INVENTION
Essential heavy metals, such as copper and zinc, are required as cofactors in redox reactions and ligand interactions and they also participate in charge stabilization, water ionization, and charge shielding during biocatalysis (Voet and Voet, 1995, In: Biochemistry, 2nd ed., John Wiley & Sons, Inc., New York). In addition to the essential heavy metals, non-essential heavy metals, such as arsenic, cadmium, mercury and lead,.are found in natural mineral deposits or as a result of human activity and they are frequently encountered by living organisms (Nriagu and Pacyna, 1988, Nature 333:134-138). Both essential and non-essential heavy metals can pose an acute problem for all living organisms in that the organisms often encounter supraoptimal concentrations of essential heavy metals and excess micromolar concentrations of non-essential heavy metals such as As, Cd, Hg and Pb. High concentrations of these non-essential heavy metals exert toxic effects through the displacement of endogenous metal cofactors, heavy or otherwise, from their cellular binding sites, aberrant reactions with the thiol groups of proteins and coenzymes, and the promotion of the formation of active oxygen species (AOS; Stadtman, 1990, Free Radic. Biol. Med. 9:315-325).
Massive global expansion in industrial and mining activities during the last two decades combined with changes in agricultural practices, have markedly increased contamination of groundwaters and soils with heavy metals. Indeed, it is estimated that the annual toxicity of metal emissions exceeds that of organics and radionuclides combined (Nriagu and Pacyna, 1988, Nature 333:134-138). Since soil and water contamination results in the uptake of heavy metals and toxins by crop plants, and eventually by humans, there is a pressing need for environmental cleanup to prevent entry of non-essential heavy metals into the food chain.
As sessile photosynthetic organisms, the mechanisms deployed by vascular plants for abrogating or alleviating heavy metal toxicity are of general interest. Not only does their lack of specialized excretory organs subject plants to large fluctuations in the levels of these substances and necessitate stringent intracellular homeostatic mechanisms, but the special status of plants as the principal points of entry of these substances into the food chain means that the mechanisms by which plants dispose of or sequester heavy metals have repercussions for all heterotrophic organisms.
In addition, bioremediation, the use of plants or microbes for the extraction and/or degradation of xenobiotics for environmental cleanup, is attracting increasing interest because of its potentially low cost by comparison with conventional physical and chemical methods. In the case of pollutants, such as heavy metals that cannot be degraded, phytoremediation is particularly appealing because of the ease with which plants can be harvested. Therefore, there is currently a great interest in the identification of native plant species or in the identification of genes from model systems useful for engineering crop species for increased resistance to and/or increased accumulation of heavy metals. In the latter category is the search for new heavy metal-binding peptides for the purpose of better understanding the mechanisms underlying the alleviation of heavy metal stress by plants and of obtaining genes encoding such peptides or the enzymes responsible for their synthesis. The identification and characterization of these genes will facilitate the development of a “mix-and-match” approach to the manipulation of plant heavy metal responses according to the specific requirements of the type of environmental site that is to be phytoremediated.
To date, three classes of peptides have been shown to contribute to heavy metal resistance in plants: glutathione (GSH), metallothioneins (MTs), and phytochelatins (PCs). The thiol peptide, GSH (&ggr;-Glu-Cys-Gly), and in some species its variant homoglutathione (h-GSH, &ggr;-Glu-Cys-&bgr;-Ala), is considered to influence the form and toxicity of heavy metals, such as As, Cd, Cu, Hg, and Zn, in several ways. These include the following mechanisms: direct metal binding (Fuhr and Rabenstein, 1973, J. Am. Chem. Soc. 95:6944-6950), promotion of the transfer of metals to other ligands (e.g, PCs and MTs; Freedman et al., 1989, J. Biol. Chem. 264:5598-5605), provision of reducing equivalents for the generation of metal oxidation states more amenable to binding by MTs and possibly PCs (Freedman et al., supra), removal of the active oxygen species formed as a result of exposure of cells to heavy metals (Inze and Van Montagu, 1995, Current Opinion in Biotech. 6:153-158), the formation of transport-active metal complexes with GSH (Li et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:42-47), and by serving as a precursor for the biosynthesis of PCs and other cysteinyl peptides (Grill et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:439-443; Meuwly et al., 1995, Plant J. 7:391-400).
The MTs, another class of peptides previously implicated in heavy metal metabolism in plants, are small (4-8 kDa), cysteine-rich metal-binding polypeptides which are induced in cells by the presence of heavy metals. MTs, which contain multiple Cys-Xaa-Cys motifs, confer tolerance to a broad range of metals in mammals (Hamer, 1986, Annu. Rev. Biochem. 55:913-951) but appear to be involved primarily in Cu homeostasis in plants (Zhou and Goldsbrough, 1994, Plant Cell 6:875-884). Arabidopsis MT1 and MT2 confer tolerance to high levels of Cu
2+
but only to low levels of Cd
2+
when heterologously expressed in MT-deficient cup1 &Dgr; mutants of
S. cerevisiae
(Zhou and Goldsbrough, 1994, Plant Cell 6:875-884). Further, MT expression in Arabidopsis seedlings is strongly induced by Cu
2+
but not by Cd
2+
(Zhou and Goldsbrough, 1994, Plant Cell 6:875-884; Murphy et al., 1997, Plant Physiol. 113:1291-1301), and comparisons between Arabidopsis ecotypes (i.e., subspecific forms in a true species, resulting from selection within a particular habitat, which can interbreed with other members of the species) demonstrate MT2 expression to be more closely correlated with Cu-tolerance than tolerance to other metals (Murphy and Taiz, 1995, Plant Physiol. 109:945-954).
Exposure of plants to heavy metals elicits the elaboration of PCs, a class peptides that play a pivotal role in heavy metal tolerance, primarily tolerance to Cd
2+
, in plants and fungi by chelating these substances and decreasing their free concentrations. PCs consist of repeating units of &ggr;-glutamylcysteine followed by a C-terminal glycine {poly-(&ggr;-Glu-Cys)
n
-Gly polymer} (Rauser, 1990, Annu. Rev. Biochem. 59:61-86; Steffens, 1990, Annu. Rev. Plant Physiol. Plant Mol. Biol. 41: 553-575). Unlike MTs, PCs are synthesized posttranslationally from GSH (&ggr;-Glu-Cys-Gly) by PC synthases (i.e., &ggr;-glutamylcysteine dipeptidyl transpeptidases, EC 2.3.2.15), which transfer a &ggr;-glutamylcysteine moiety from GSH to a second molecule or a previously synthesized PC molecule (Rauser, 1990, Annu. Rev. Biochem. 59:61-86; Zenk, 1996, Gene 179:21-30). Found in some fungi and in all plant species investigated to date (Rauser, 1990, Annu. Rev. Biochem. 59:61-86; Zenk, 1996, Gene 179:21-30), PCs bind heavy metals, such as Cd
2+
, with high affinity, and localize together with Cd
2+
to the vacuole of intact cells (Vogeli-Lange and Wagner, 1990, Plant Physiol. 92:1086-1093). As indicated by the hypersensitivity of PC-deficient Arabidopsis cadl mutants to Cd
2+
but not to Cu
2+
(Howden et al., 1995, Plant Physiol. 107:1059-1066), PCs contribute most markedly to Cd
2+
detoxification in planta. PC-dependent vacuolar Cd
2+
sequestration is best understood in the fission yeast
Schizosaccharomyces pombe,
in which the hmt1
+
gene product, a PC-selective ATP-binding cassette (ABC) transporter, pumps Cd.PCs and apo-PCs from the cytosol into the vacuole at the expense of ATP (Ortiz et al., 1992, EMBO J. 11:3491-3499; Ort

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