Molecular, cellular and physiological characterization of amino acid transporters in Arabidopsis thaliana

The acquisition and partitioning of amino acids is essential for plant growth. Translocation of amino acids from cell to cell and long-distance is predicted to be by transporters located in the plasma membrane of plant cells. This research focuses on the Arabidopsis thaliana amino acid transporters AtAAP1, AtAAP2, and AtLHT2 and their molecular, cellular and physiological characterization to resolve transporter function in planta. Localization studies revealed that AtAAP1 is functioning in the plasma membrane of root epidermal cells. To analyze the importance of AtAAP1 in acquisition of amino acids from the soil aap1 mutants were tested for altered phenotype. Growth of seedlings on toxic concentrations of amino acids and uptake studies with radiolabeled amino acids showed difference in acquisition between aap1 and wild-type. The studies revealed that AtAAP1 is important for uptake of glutamate and neutral amino acids by the roots. Following acquisition from the soil or assimilation from inorganic nitrogen in roots or leaves, amino acids are transported long-distance through the phloem to developing sink organs like flowers. Using green fluorescence protein-transporter protein fusions the amino acid permease AtAAP2 was localized to the phloem throughout Arabidopsis demonstrating its function in phloem loading. Interestingly, AtAAP2 was localized to companion cells (CC) as well as sieve elements (SE) of the phloem, although in mature SEs the transcriptional/translational apparatus is absent. This strongly suggests that AtAAP2 protein is trafficking through plasmodesmata from the CCs to the SEs and that phloem loading with amino acids occurs via both CCs and SEs. Amino acids arriving via the phloem in sink organs are unloaded and then distributed within the sink for growth and development. In flowers, the newly identified AtLHT2 is specifically expressed in the anther tapetum where AtLHT2 is predicted to have a critical function in supplying the tapetum cells with amino acids for synthesis of compounds important for microspore structure and in transfer of organic nitrogen to the locule for pollen development. Biochemical analysis in Saccharomyces cerevisiae mutants revealed that AtLHT2 represents a novel high-affinity transport system for acidic and neutral amino acids.