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Metabolic engineering approaches are increasingly employed for environmental applications. Because phytochelatins (PC) protect plants from heavy metal toxicity, strategies directed at manipulating the biosynthesis of these peptides hold promise for the remediation of soils and groundwaters contaminated with heavy metals. Directed evolution of Arabidopsis thaliana phytochelatin synthase (AtPCS1) yields mutants that confer levels of cadmium tolerance and accumulation greater than expression of the wild-type enzyme in Saccharomyces cerevisiae, Arabidopsis, or Brassica juncea. Surprisingly, the AtPCS1 mutants that enhance cadmium tolerance and accumulation are catalytically less efficient than wild-type enzyme. Metabolite analyses indicate that transformation with AtPCS1, but not with the mutant variants, decreases the levels of the PC precursors, glutathione and γ-glutamylcysteine, upon exposure to cadmium. Selection of AtPCS1 variants with diminished catalytic activity alleviates depletion of these metabolites, which maintains redox homeostasis while supporting PC synthesis during cadmium exposure. These results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools available for environmental remediation. Background: Plants synthesize phytochelatin peptides for protection against heavy metals. Results: Metabolic engineering in yeast and plants using a phytochelatin synthase variant leads to improved cadmium tolerance. Conclusion: Enhanced cadmium tolerance results from a balance between phytochelatin synthesis and redox state. Significance: Our results emphasize the importance of metabolic context for pathway engineering and broaden the range of tools for environmental remediation.

Arsenic is an extremely toxic metalloid causing serious health problems. In Southeast Asia, aquifers providing drinking and agricultural water for tens of millions of people are contaminated with arsenic. To reduce nutritional arsenic intake through the consumption of contaminated plants, identification of the mechanisms for arsenic accumulation and detoxification in plants is a prerequisite. Phytochelatins (PCs) are glutathione-derived peptides that chelate heavy metals and metalloids such as arsenic, thereby functioning as the first step in their detoxification. Plant vacuoles act as final detoxification stores for heavy metals and arsenic. The essential PC–metal(loid) transporters that sequester toxic metal(loid)s in plant vacuoles have long been sought but remain unidentified in plants. Here we show that in the absence of two ABCC-type transporters, AtABCC1 and AtABCC2, Arabidopsis thaliana is extremely sensitive to arsenic and arsenic-based herbicides. Heterologous expression of these ABCC transporters in phytochelatin-producing Saccharomyces cerevisiae enhanced arsenic tolerance and accumulation. Furthermore, membrane vesicles isolated from these yeasts exhibited a pronounced arsenite [As(III)]–PC 2 transport activity. Vacuoles isolated from atabcc1 atabcc2 double knockout plants exhibited a very low residual As(III)–PC 2 transport activity, and interestingly, less PC was produced in mutant plants when exposed to arsenic. Overexpression of AtPCS1 and AtABCC1 resulted in plants exhibiting increased arsenic tolerance. Our findings demonstrate that AtABCC1 and AtABCC2 are the long-sought and major vacuolar PC transporters. Modulation of vacuolar PC transporters in other plants may allow engineering of plants suited either for phytoremediation or reduced accumulation of arsenic in edible organs.

The ABC superfamily comprises both membrane-bound transporters and soluble proteins involved in a broad range of processes, many of which are of considerable agricultural, biotechnological and medical potential. Completion of the Arabidopsis and rice genome sequences has revealed a particularly large and diverse complement of plant ABC proteins in comparison with other organisms. Forward and reverse genetics, together with heterologous expression, have uncovered many novel roles for plant ABC proteins, but this progress has been accompanied by a confusing proliferation of names for plant ABC genes and their products. A consolidated nomenclature will provide much-needed clarity and a framework for future research.

1,1-Diphosphonate analogs of pyrophosphate, containing an amino or a hydroxyl group on the bridge carbon atom, are potent inhibitors of the H(+)-translocating pyrophosphatases of chromatophores prepared from the bacterium Rhodospirillum rubrum and vacuolar membrane vesicles prepared from the plant Vigna radiata. The inhibition constant for aminomethylenediphosphonate, which binds competitively with respect to substrate, is below 2 microM. Rat liver mitochondrial pyrophosphatase is two orders of magnitude less sensitive to this compound but extremely sensitive to imidodiphosphate. By contrast, fluoride is highly effective only against the mitochondrial pyrophosphatase. It is concluded that the mitochondrial pyrophosphatase and the H(+)-pyrophosphatases of chromatophores and vacuolar membranes belong to two different classes of enzyme.

Previous work with membrane visicles has demostrated an absolute dependence on K+ for proton translocation by the inorganic pyrophosphatase (H+-PPase: EC 3.6.1.1) from the vacuolar membrane (tonoplast) of higher plants. Using intact vacuoles from sugar beel (Beta vulgaris) storage tissue, we have monitored PP,-dependent currents by patch clamp in ‘whole vacuole’ mode. Serial K+ substitutions were made are both tonoplast faces. The results show that K+ activation occurs only at the cytosolic face.