Development of a new analytical tool for elucidating nitrate signaling networks

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This project aimed to develop a new fluorescent indicator of changes in nitrate concentration within living plant roots. This indicator relies on Forster Resonance Energy Transfer (FRET) between two fluorescent proteins, such that a change in nitrate level is shown by a ratio change in emission from the two proteins.Synechococcus PCC 7924 cyanobacterium contains a high-affinity nitrate binding protein, NrtA, and the gene encoding this protein was used for the new indicator. After the NrtA was cloned into the appropriate vectors, the gene was fused between genes encoding CFP and YFP. The new fusion protein successfully showed FRET activity, and dual emission from both CFP and YFP when only the CFP protein was excited. A slight increase in the FRET ratio was also observed when the protein was exposed to higher nitrate concentrations. However, a high FRET ratio was present even at low nitrate levels, and the ratio increase was not enough for use in plants. Conserved residues in NrtA were identified and modified using site-directed mutagenesis. This successfully reduced the high initial FRET ratio but did not result in a greater nitrate response. Further optimization is expected to provide the new practical indicator for plant research.