ARS5 is a component of the 26S proteasome complex, and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis.

A forward-genetic screen in Arabidopsis led to the isolation of several arsenic tolerance mutants. ars5 was the strongest arsenate- and arsenite-resistant mutant identified in this genetic screen. Here, we report the characterization and cloning of the ars5 mutant gene. ars5 is shown to exhibit an increased accumulation of arsenic and thiol compounds during arsenic stress. Rough mapping together with microarray-based expression mapping identified the ars5 mutation in the alpha subunit F (PAF1) of the 26S proteasome complex. Characterization of an independent paf1 T-DNA insertion allele and complementation by PAF1 confirmed that paf1 mutation is responsible for the enhanced thiol accumulation and arsenic tolerance phenotypes. Arsenic tolerance was not observed in a knock-out mutant of the highly homologous PAF2 gene. However, genetic complementation of ars5 by the overexpression of PAF2 suggests that the PAF2 protein is functionally equivalent to PAF1 when expressed at high levels. No detectible difference was observed in total ubiquitinylated protein profiles between ars5 and wild-type (WT) Arabidopsis, suggesting that the arsenic tolerance observed in ars5 is not derived from a general impairment in proteasome-mediated protein degradation. Quantitative RT-PCR showed that arsenic induces the enhanced transcriptional activation of several key genes that function in glutathione and phytochelatin biosynthesis in the WT, and this arsenic induction of gene expression is more dramatic in ars5. The enhanced transcriptional response to arsenic and the increased accumulation of thiol compounds in ars5, compared with WT, suggest the presence of a positive regulation pathway for thiol biosynthesis that is enhanced in the ars5 background.

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