Asparagine metabolism and function in nitrogen signaling

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The amino acid asparagine is a major form of nitrogen commonly transported to sink tissues of higher plants, including roots, flowers and developing seeds, due to its high nitrogen to carbon ratio. Local biosynthesis of asparagine in reproductive tissues can also pre-condition storage reserve accumulation in grain. In leaf, asparagine is accumulated transiently at night when, in the absence of photosynthesis, levels of organic acids are insufficient for nitrogen assimilation. Asparagine is an important intermediate in photorespiration, providing an input of nitrogen to balance an output of serine or glycine used in other pathways. The objectives of this proposal are to achieve a detailed understanding of asparagine metabolism and of the function and perception of asparagine as a metabolite signal of organic nitrogen in higher plants. Reporter screens with previously identified asparagine-responsive genes will help identify factors involved in their regulation. Gene regulatory networks will also be investigated by in silico promoter analysis, identification of cognate regulators and inducible gene profiling assays. The biochemistry of the photorespiratory pathway of asparagine metabolism will be investigated. The enzyme omega-amidase is involved in the breakdown of the reaction product of asparagine transamination, alpha-ketosuccinamate and of its reduced form, alpha-hydroxysuccinamate. It shares a high degree of sequence identity with an enzyme catalyzing the breakdown of a damaged form of the antioxidant glutathione, arising from transamination. The structural determinants of substrate specificity in these enzymes will be identified by an approach combining protein crystallography and site-directed mutagenesis. The dehydrogenase enzyme responsible for the reduction of alpha-ketosuccinamate will be identified. Two alternative approaches will be used. First, a bioinformatic approach will examine co-expression of asparagine metabolic and photorespiratory genes with dehydrogenases in the genome of the model plant Arabidopsis thaliana. Candidate genes will be characterized by determining the catalytic activity of the corresponding recombinant enzyme in vitro. The second approach will involve purification of the enzyme from soybean leaves, and identification of the purified protein by digestion with a site-specific protease, mass spectrometry and search against a proteome database. Candidate genes will be validated by profiling metabolites in the corresponding knock-out mutant of A. thaliana in response to treatment with exogenous non-labeled and stably labeled asparagine. The research results will improve our understanding of asparagine metabolism and signaling in higher plants. **