Genetically encoded sensors for ions and metabolites

Many of the genes encoding important trans porters and metabolic enzymes have been identified over the last ten years. Moreover it has been possible to study the biochemical properties of the corresponding proteins in great detail. It is expected that by 2,010 biochemical functions will have been assigned to many of the products of the approximately 30,000 Arabidopsis genes. We will get closer to understanding the biological function of the gene products by systematic analysis of mutants using knock-out and TILLING approaches. Metabolomics initiatives complement these approaches by providing insight into the changes in cellular ion and metabolite profiles in the mutants, thus giving information essential for the construction of cellular and whole plant models. However, one important dataset especially relevant to multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of ions and metabolite levels at cellular and subcell ular levels. To address this issue, we have developed protein-based nanosensors for several metabolites, providing a set of tools for the determination of cytosolic and subcellular ion (e.g. iron and zinc) and metabolite levels in real time using fluorescence-based microscopy. The prototypes of these sensors were shown to function in vitro and also in vivo, i.e. in yeast and in mammalian cell cultures. One future goal is to expand the set of sensors to a wider spectrum of targets by using the natural spectrum of periplasmic binding proteins from bacteria and by computational design of proteins with altered binding pockets. Application of nanosensor technology to plant cells and tissues will help to elucidate the special and temporal distribution of ions and metabolites.