Soil contamination with arsenic (As) can cause phytotoxicity and elevated As accumulation in rice grain. Here, we used a forward genetics approach to investigate the mechanism of arsenate (As(V)) tolerance and accumulation in rice. A rice mutant hypersensitive to As(V), but not to As(III), was isolated. Genomic resequencing and complementation tests were used to identify the causal gene. The function of the gene, its expression pattern and subcellular localization were characterized. OsHAC4 is the causal gene for the As(V)-hypersensitive phenotype. The gene encodes a rhodanase-like protein that shows As(V) reductase activity when expressed in Escherichia coli. OsHAC4 was highly expressed in roots and was induced by As(V). In OsHAC4pro-GUS transgenic plants, the gene was expressed exclusively in the root epidermis and exodermis. OsHAC4-eGFP was localized in the cytoplasm and the nucleus. Mutation in OsHAC4 resulted in decreased As(V) reduction in roots, decreased As(III) efflux to the external medium and markedly increased As accumulation in rice shoots. Overexpression of OsHAC4 increased As(V) tolerance and decreased As accumulation in rice plants. OsHAC4 is an As(V) reductase that is critical for As(V) detoxification and for the control of As accumulation in rice. As(V) reduction, followed by As(III) efflux, is an important mechanism of As(V) detoxification.

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SummaryArsenic (As) contamination in a paddy environment can cause phytotoxicity and elevated As accumulation in rice (Oryza sativa). The mechanism of As detoxification in rice is still poorly understood.We isolated an arsenate (As(V))-sensitive mutant of rice. Genomic resequencing and complementation identified OsCLT1, encoding a CRT-like transporter, as the causal gene for the mutant phenotype.OsCLT1 is localized to the envelope membrane of plastids. The glutathione and cglutamylcysteine contents in roots of Osclt1 and RNA interference lines were decreased markedly compared with the wild-type (WT). The concentrations of phytochelatin PC 2 in Osclt1 roots were only 32% and 12% of that in WT after As(V) and As(III) treatments, respectively. OsCLT1 mutation resulted in lower As accumulation in roots but higher As accumulation in shoots when exposed to As(V). Under As(III) treatment, Osclt1 accumulated a lower As concentration in roots but similar As concentration in shoots to WT. Further analysis showed that the reduction of As(V) to As(III) was decreased in Osclt1. Osclt1 was also hypersensitive to cadmium (Cd).These results indicate that OsCLT1 plays an important role in glutathione homeostasis, probably by mediating the export of c-glutamylcysteine and glutathione from plastids to the cytoplasm, which in turn affects As and Cd detoxification in rice.

Phosphate (Pi) uptake in plants depends on plasma membrane (PM)-localized phosphate transporters (PTs). OsCK2 phosphorylates PTs and inhibits their trafficking from the endoplasmic reticulum (ER) to the PM in rice (Oryza sativa), but how PTs are dephosphorylated is unknown. We demonstrate that the protein phosphatase type 2C (PP2C) protein phosphatase OsPP95 interacts with OsPT2 and OsPT8 and dephosphorylates OsPT8 at Ser-517. Rice plants overexpressing OsPP95 reduced OsPT8 phosphorylation and promoted OsPT2 and OsPT8 trafficking from the ER to the PM, resulting in Pi accumulation. Under Pi-sufficient conditions, Pi levels were lower in young leaves and higher in old leaves in ospp95 mutants than in those of the wild type, even though the overall shoot Pi levels were the same in the mutant and the wild type. In the wild type, OsPP95 accumulated under Pi starvation but was rapidly degraded under Pi-sufficient conditions. We show that OsPHO2 interacts with and induces the degradation of OsPP95. We conclude that OsPP95, a protein phosphatase negatively regulated by OsPHO2, positively regulates Pi homeostasis and remobilization by dephosphorylating PTs and affecting their trafficking to the PM, a reversible process required for adaptation to variable Pi conditions.