Studies on two phytochelatin transporters important for metal/metalloid tolerance and accumulation in plant

DoctorPlants are exposed to various potentially toxic compounds including heavy metals and toxic metalloids. Intake of crop plants accumulating excess metal(loid)s provides a major route for the entry of toxic ions into the human body. Prolonged exposure to metal(loid)s such as arsenic, cadmium and mercury can cause serious health problemsfor example, arsenic poisoning increases the risk of bladder cancer development, while cadmium and mercury are known as the causes of Itai-Itai disease and Minamata disease, respectively. To reduce nutritional metal(loid) absorption through plants, elucidation of the accumulation and detoxification mechanisms for toxic metal(loid)s in plants is a prerequisite. Heavy metal and metalloid detoxification mechanism is mediated by two major steps in plant, chelation by phytochelatins (PCs) and subsequent transport of the PC-metal(loid) complexes into a storage organelle, vacuole. PCs are peptide-type chelators, which bind to heavy metals and metalloids specifically, functioning as the first step in their detoxification. Plant vacuoles act as final detoxification stores for heavy metals and metalloids, and their vacuolar sequestration is mediated by active transporters such as ABC transporters. The essential transporters implicated in this process have been long-sought, but remained unidentified in plants. The aim of this work was to find a PC transporter which is involved in metal(loid) detoxification in plants. For this purpose, I screened and isolated Arabidopsis mutants of ABC transporters, which exhibited altered tolerance to excess of arsenicals and cadmium. Here I report about the evidence which supports that two ABC transporters, AtABCC1 and AtABCC2 function in vacuolar transport of PC-metal(loid) complex, and their importance in detoxification of arsenic, cadmium and mercury. In the study elucidating their role in arsenic detoxification, I found 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 transporters in phytochelatin-producing Saccharomyces cerevisiae enhanced arsenic tolerance and accumulation. Furthermore, membrane vesicles isolated from the yeasts exhibited a pronounced As(III)-PC2 transport activity. Vacuoles isolated from atabcc1 atabcc2 double knockout plants exhibited a very low residual As(III)-PC2 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. These findings demonstrate that AtABCC1 and AtABCC2 are the major vacuolar PC transporters for arsenic detoxification in Arabidopsis. In the second part describing the roles of AtABCC1 and AtABCC2 in Cd and Hg(II) detoxification, I found out that atabcc1 single or atabcc1 and atabcc2 double knock-out mutant plants exhibited a hypersensitive phenotype in the presence of Cd(II) and Hg(II). Microscopic analysis using a Cd sensitive probe revealed that Cd is located mostly in the cytosol in protoplasts of the double mutant, unlike in wild-type cells where it was detected mainly in the vacuole, suggesting that the two ABC transporters are important for vacuolar sequestration of Cd. Heterologous expression of the two ABC transporters in yeast confirmed their importance in heavy metal tolerance. Overexpression of AtABCC1 in Arabidopsis resulted in enhanced Cd(II) tolerance and accumulation. Together, it can be concluded that AtABCC1 and AtABCC2 are important vacuolar transporters, which confer tolerance to cadmium and mercury. In summary, I demonstrated that the two PC transporters play important roles in detoxification and accumulation of three metal(loid)s, arsenic, cadmium and mercury in plant. These findings will contribute to understand the basis of heavy metal detoxification mechanisms, which are important for optimal growth of plant under metal(loid) stress conditions. Moreover, modulation of the vacuolar transporters may allow engineering plants suited either for phytoremediation or reduced accumulation of toxic ions in edible organs