Using a chemical genetics approach to dissect the nitrogen signalling pathway in Arabidopsis

Nitrate is an important nutrient and signalling molecule to plants. As it is taken up and assimilated, reduced forms of N accumulate and the expression of many genes associated with nitrate assimilation are repressed. Little is known about the mechanisms involved in this N repression. This project, for the first time, adopts a chemical genetics approach to investigate the feedback regulatory pathway that links the plant’s N status to expression of the NRT2.1 nitrate transporter gene. A novel chemical screening platform was developed that was designed to be used in conjunction with Arabidopsis lines expressing luciferase reporter genes in roots. This semi-hydroponic platform allows roots to be exposed to a variety of nutrient treatments in a 96-well plate format suitable for chemical genetic screens. This was combined with a newly developed ‘ice capture’ method that provided a rapid and efficient way to harvest root material for the luciferase assay. Using this screening platform in conjunction with a nitrate-inducible luciferase reporter line, pNRT2.1::LUC, three chemical libraries, containing 7420 bioactive molecules were screened in duplicate for compounds that antagonise N repression of luminescence. The screen identified a plant-derived alkaloid, camptothecin, that enhanced pNRT2.1::LUC expression under N-repressive conditions. The positive effect of camptothecin on expression of the endogenous NRT2.1 gene was confirmed using real-time PCR and shown to extend to other N-repressed genes of the nitrate assimilatory pathway. Camptothecin is known to target topoisomerase I, an enzyme that is increasingly being linked to a role in chromatin re-modelling, in addition to its more familiar roles in DNA replication and repair. The possible epigenetic role of topoisomerase I in repression of NRT2.1 and other genes of the nitrate assimilatory pathway is discussed. It was also observed that an arginine treatment strongly stimulated pNRT2.1::LUC in the luciferase assay, in a nitrate-dependent manner. Since this effect was not observed at the mRNA level, it is hypothesised that arginine was acting on pNRT2.1 expression at a post-transcriptional level.