Nitrogen-status sensing in Arabidopsis

Plant nitrogen use efficiency (NUE) is important in agriculture because it significantly influences yield and fertiliser requirements. Many factors influence NUE; these include nitrogen availability, internal nitrogen-status, nitrogen uptake and assimilation rates as well as carbon status. Previous attempts to improve NUE by targeting the main nitrogen uptake or assimilation machinery (e.g. over-expression of nitrate reductase or glutamine synthetase) have had mixed results. This is because these enzymes are strictly controlled by multiple feedback mechanisms relating to internal and external nitrogen-status. Arguably, a better approach to manipulating NUE would be to identify and supress these feedback signals either alone, or in conjunction with over-expression of the uptake and assimilation machinery. This could enhance exploitation of nitrogen resources leading to increased growth. Internal nitrogen-status signalling is poorly understood. However, it is known that amino acids, when exogenously supplied, inhibit the uptake of nitrate. The mechanism for this inhibition has not been identified, but it is likely the result of sensing of internal nitrogen status, the amino acids representing a signal of nitrogen sufficiency, in turn leading to reduced expression and activity of the nitrogen uptake and assimilation machinery. In this thesis I conducted a reverse-genetic screen based on the response of candidate amino acid transporter knock-out mutants to exogenous alanine. In wild-type Arabidopsis, exogenous alanine caused inhibition of vegetative growth, which is accompanied by a reduction in nitrate uptake and transcripts encoding nitrate transporters. In the reverse-genetic growth screen, I identified four mutants with vegetative growth that was insensitive to exogenous alanine. Further characterisation of these mutants revealed that nitrogen uptake was not inhibited and this was accompanied by a lack of repression of nitrate transporter transcripts. Based on this evidence, I concluded that these amino acid transporter mutants were perturbed in nitrogen-status signalling. However, the origin of this signalling perturbation remains unclear. I formulated two hypotheses to explain how four amino acid transporter mutants could have the same signalling phenotype. The first suggested that the four alanine-insensitive transporters could form a functional complex, which is part of the nitrogen-status signalling pathway. The second suggested that the loss-of-function of the amino acid transporter perturbs metabolite levels, in a common way in each mutant, producing a metabolic signature which is sensed downstream of the transporter by an unknown mechanism. The latter hypothesis was tested by extensive metabolite profiling and I could find no evidence in support of the hypothesis. Preliminary investigations were made of the functional-complex hypothesis but technical problems prevented significant progress.