Elucidating the signalling pathway involving ANR1

Nitrogen is considered to be often the limiting factor for plant growth in natural ecosystems. The environmental problems linked with usage of nitrate-containing fertilizer in agricultural systems to fulfil the plants demand for nitrogen are well known. Understanding of nitrate utilization at the molecular level could be taken into advantage for a more efficient nitrate uptake in agricultural systems. The molecular processes in nitrate assimilation have been well studied in the fungus Aspergillus nidulans. In plants nitrate utilization has been widely studied at the physiological level and, more recently, also with increasing effort at the molecular level. The major enzymes involved in nitrate assimilation, such as nitrate transporter and nitrate reductase, are highly conserved between fungi and plants. The regulation of nitrate assimilation in plants is more complex, but recent advances in the Arabidopsis research revealed that there are similarities in the regulation of nitrate responses at the molecular level. The ANR1 (for Arabidopsis Nitrate Regulated) MADS-box transcription factor was the first transcription factor in plants found to be activated by the NO 3 - ion, which is in analogy with the NirA transcription factor which is also activated by nitrate. Deletions of nirA and ANR1, respectively, lead both to defects in the nitrate uptake system beside other organism-specific defects in response to nitrate. During my work with the nitrate assimilation specific transcription factor NirA in Aspergillus nidulans it became clear that nuclear translocation of NirA is a key step in activation of the nitrate assimilation genes. This nuclear translocation of NirA is specifically induced by the presence of NO 3 - in the environment. In Arabidopsis thaliana the transcription factor ANR1 is a component of a signalling pathway, that is induced by the NO 3 - ion. It has been shown that Arabidopsis lines with deletions of ANR1 are defective in their response to localized NO 3 - supply, they lost the ability to stimulate lateral root growth. The role of ANR1 in nitrate signalling seems to be that of a master regulator of the plants response to fluctuating supplies of NO 3 - . There is a strong evidence that ANR1 is activated by posttranslational modifications, which induce its nuclear translocation, where it binds to promoter regions of its target genes. However, this hypothesis has to be verified by molecular studies. My contribution in verification of this hypothesis will be to construct an ANR1-GFP fusion and to determine the subcellular localisation of ANR1. Performing CLSM (confocal laser scanning microscopy), the GFP-tagged ANR1 will be investigated whether its nuclear localization is triggered by a NO 3 - signal. Furthermore, I will make a yeast two hybrid screen for identification of other components involved in the same signalling pathway as ANR1. It has been shown for other members of the MADS-box family in Arabidopsis, that their nuclear translocation is triggered by complex formation with other proteins and/or by phosphorylation. The proposed studies with ANR1 will bring light into understanding the molecular processes induced by nitrate. The knowledge of these molecular processes could provide solutions for a more efficient usage of nitrate supplies in agricultural systems.