The Arabidopsis J-protein DJA4 protects chloroplasts from arsenic toxicity by binding arsenite

Chloroplasts are a sensitive target of arsenic (As) toxicity. How chloroplasts are protected against As toxicity remains unclear. We isolated an As-hypersensitive Arabidopsis thaliana mutant, aic2 cad1-3, from an ethyl methanesulfonate-mutagenized cad1-3 background defective in phytochelatin synthase. This double mutant displayed enhanced leaf chlorosis, reduced chloroplast number, impaired chloroplast structure, and larger growth inhibition under As stress compared with cad1-3. Map-based cloning identified the causal gene for the mutant phenotype as DJA4 belonging to the J-protein family. DJA4 protein was localized to the chloroplast stroma. The aic2 single mutant and DJA4 knockout mutants were also more sensitive to As than wild-type. Microscale thermophoresis analysis showed that the recombinant DJA4^62-403 protein binds arsenite. Genetic analyses demonstrated that double knockout of DJA4 and TOC132 (Translocon at the Outer envelope membrane of Chloroplasts) resulted in greater As sensitivity compared with single-gene knockouts. Chloroplast proteomic analysis identified nine proteins commonly altered in both dja4 and toc132 mutants under As stress, many of which are involved in chloroplast metabolism and redox homeostasis. These results show that DJA4 plays a crucial role in maintaining chloroplast function under As stress by binding with arsenite and protecting the chloroplast protein import machinery.