Minisymposium 28: Biotechnology

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Abs # M2803: Arabidopsis arsenate reductase (AtACR2): a single enzyme wity multiple functions

Presenter:	Dhankher, Om Parkash       Contact Presenter
Authors	Dhankher, Om Parkash (A)
Affiliations:	(A): University of Massachusetts Amherst

Arsenic (As) contaminated soils and water supplies are major sources of food chain contamination and thereby endanger human health. Arsenate (As^V) is a phosphate analog, has been shown to be taken up via the phosphate uptake system and further, most of the As in roots gets reduced to arsenite (As^III) endogenously (Dhankher et al., 2002, Nature Biotech. 20:1140-45). However, the exact mechanism of As^V reduction and long distance transport of As^V or As^III from roots to shoot tissues and further detoxification in plants is not known. Recently, we have identified an endogenous arsenate reductase, AtACR2, from Arabidopsis that reduces As^V to As^III in plants. The AtACR2 has a catalytic domain, HCX5R, required for arsenate reductase activity and a C-terminal cysteine-rich metal-binding domain. The AtACR2 complemented the function of arsenate reductase in E. coli strains deficient in arsenate reductase ArsC. We knocked down the ACR2 expression using RNAi approach in Arabidopsis as low as 2% of wild-type levels and the transgenic lines were more sensitive to As^V but not to As^III. Inactivation of AtACR2 by RNAi caused the translocation of 10-16 fold more As from root to shoot tissues when these plants were exposed to As^V, but not when they were exposed to As^III (Dhankher et al., 2006, PNAS 103: 5413-18). In addition, the ACR2 knockdown plants accumulated 2- to 3-fold less phosphorus in shoots compared with wild-type in response to As^V exposure. All these compelling evidences suggest that Arabidopsis ACR2 encodes a functional arsenate reductase. Arabidopsis ACR2 sequences show homology to a region within the CDC25 superfamily of protein-tyrosine phosphatases (PTPases) that also contains the conserved HCX5R domain and has recently been shown to act as PTPase: functioning in vitro by activating plant cyclin-dependent kinase activity and hydrolyzing phosphate from artificial substrates (Landrieu et al., 2004, PNAS 101: 13380-85). We replaced conserved cysteine residues with serine in the catalytic domain (C72S) and in the C-terminal metal-binding domain (C122, 1227S). Our preliminary results showed that Arabidopsis plants overexpressing the mutated AtACR2_c72s and AtACR2_c122,127s lost resistance to arsenate and grew similar to wild-type controls on media containing various levels of arsenate. These studies clearly suggest that AtACR2 has multiple functions in plants and altering its expression in plants will play a vital role in the phytoremediation of As contamination.