| Amino acids are the main transport form of organic nitrogen in plants and their import into plant cells is predicted to be by plasma membrane transport proteins. This research analyzed the roles of the amino permease AtAAP1 as well as AtLHT amino acid transporters in Arabidopsis with an emphasis on amino acid uptake by the roots and transport within flowers. Expression of AtAAP1 and AtLHT6 was localized to root cells, including root hairs. Furthermore, the transporters were targeted to the plasma membrane, indicating a function in nitrogen import into cells. Transport studies were performed with aap1 and lht6 seedlings and showed reduced amino acid uptake in mutants compared to wild type. Moreover, aap1 mutants could grow on media containing amino acid concentrations that are toxic to wild type, and plants overexpressing AAP1 displayed increased uptake of amino acids. Together, these data demonstrate that transporters are important for root amino acid acquisition and that genetic manipulation of root located amino acid import systems results in alteration of nitrogen uptake.When analyzing the expression of LHT amino acid transporters it was found that LHTs are strongly expressed in flowers. AtLHT2, AtLHT4, AtLHT5 , and AtLHT6 promoter-GUS studies were performed, and dependent on the transporter, GUS staining was localized in the tapetal cells of the anther, germinating pollen, pollen tubes, stigma or transmitting tissue of the pistil. Subcellular localization of AtLHT:GFP fusion proteins showed that all LHT transporters are targeted to the plasma membrane, suggesting that the LHTs function in the import of nitrogen into floral tissues, thereby supplying sufficient nutrients for reproductive success. Localization of AtLHTs expression was also determined throughout the plant using promoter-GUS studies and histochemical detection displayed tissue- and cell-specific expression patterns. Furthermore, AtLHTs were expressed in yeast cells and Xenopus oocytes to determine their substrate selectivity, but none of the LHTs showed transport function in these heterologous systems. For future studies homozygous lht mutants were identified, crosses were performed with selected mutants to produce double mutants and AtLHT4, 5 and 6 antisense lines were produced. |
