Genetic, Genomic and Biochemical Analysis of Arabidopsis Threonine Aldolase and Associated Molecular and Metabolic Networks

Since the amino acids threonine and isoleucine can be limiting in mammalian diet and there is interest in increasing their abundance in certain crop plants. To meet this need, a BARD proposal was written with two main research objectives: (i) investigate new avenues for manipulating threonine and isoleucine content in plants and (ii) study the role of threonine aldolase in plant metabolism. Research conducted to meet these goals included analysis of the sub-cellular localization of threonine aldolase in the plant, analysis of metabolic flux in developing embryos, over- and under-expression of Arabidopsis threonine aldolases, and transcriptional and metabolic analysis of perturbations resulting from altered threonine aldolase expression. Additionally, the broader metabolic effects of increasing lysine biosynthesis were investigated. An interesting observation that came up in the course of the project is that threonine aldolase activity affects methionine gamma-lyase in Arabidopsis. Further research showed that threonine deaminase and methionine gamma-lyase both contribute to isoleucine biosynthesis in plants. Therefore, isoleucine content can be altered by manipulating the expression of either or both of these enzymes. Additionally, both enzymes contribute to the up to 100-fold increase in isoleucine that is observed in drought-stressed Arabidopsis. Toward the end of the project it was discovered that through different projects, both groups had been able to independently up-regulate phenylalanine accumulation by different mechanisms. The Galili lab transformed Arabidopsis with a feedbackinsensitive bacterial enzyme and the Jander lab found a feedback insensitive mutation in Arabidopsis arogenate dehydratase. Exchange of the respective plant lines has allowed a comparative analysis of the different methods for increasing phenylalanine content and the creation of double mutants. The research that was conducted as part of this BARD project has led to new insights into plant amino acid metabolism. Additionally, new approaches that were found to increase the accumulation of threonine, isoleucine, and phenylalanine in plants have potential practical applications. Increased threonine and isoleucine levels can increase the nutritional value of crop plants. Elevated isoleucine accumulation may increase the osmotic stress tolerance of plants. Up-regulation of phenylalanine biosynthesis can be used to increase the production of downstream higher-value plant metabolites of biofuel feed stocks.