Identification and characterization of chloroplast amino acid transporters in Arabidopsis thaliana

Samantha Vivia The

doi:10.7273/000007170

Nitrogen (N) is an essential nutrient for plant growth and required in relatively high quantities. Plants generally take up N in the form of nitrate and ammonium that are assimilated into amino acids in root plastids or in chloroplasts of mature leaves. Most of the 20 proteinogenic amino acids are produced in the chloroplasts. Therefore, various transport systems are required in the plastidial envelope membrane to move amino acids from the stroma to the cytosol for cellular metabolism or for export from the leaf to supply growing sinks with N. However, despite their importance, transporters functioning in the plastidial export of amino acids remained mostly unknown. In this graduate project, three putative chloroplast transporters were identified in Arabidopsis based on the presence of chloroplast targeting peptides, chloroplast proteome data, and transporter co-expression with genes associated with N or carbon metabolism and transport in subcellular compartments. These candidates were USUALLY MULTIPLE ACID MOVE IN AND OUT TRANSPORTER 44 (UMAMIT44), L-type amino acid transporter 5 (LAT5), and tyrosine specific transporter 1 (TyrP1). The transporters were fused to green fluorescence proteins (GFP) and their localization to chloroplasts was demonstrated. This dissertation work then characterized the three transporters in more detail and resolved their function in plastidial amino acid export and importance for cellular metabolism, long-distance transport of N and overall plant physiology. First, the role of UMAMIT44 in plastidial amino acid transport was examined. Analyses of umamit44 mutants resolved that the transporter mediates glutamate export from chloroplasts and thereby regulates plastidial and extraplastidial glutamate homeostasis. The results further demonstrated that UMAMIT44 plays an important role in source leaf N and carbon (C) metabolism, as well as long-distance transport of N and C to sinks, with consequences for plant growth and development. Second, LAT5 function in chloroplasts was elucidated using two mutant lines. Metabolite analyses revealed that the transporter is important for arginine export from chloroplasts and regulates arginine levels within and outside of the plastids. It was discovered that LAT5 plays a major role in cellular functions under high levels of photorespiration. Indeed, LAT5-mediated export of arginine and its subsequent mitochondrial import were found to be essential for the safe transfer of toxic ammonia from the mitochondria to chloroplasts for reassimilation. Finally, TyrP1 function in plastidial amino acid transport was addressed. Metabolite analyses of tyrp1 mutants support that TyrP1 is involved in tyrosine export from chloroplasts and its function affects the amino acid balance in leaves. The mutants displayed reduced rosette leaf biomass and surface area and decreased seed yield, demonstrating that TyrP1 function is essential for plant growth and seed development. Overall, this dissertation work reveals that plastidial glutamate, arginine and tyrosine export is important for regulating plastidial and extraplastidial amino acid availability and that the function of the transporters impacts leaf N and C metabolism, and leaf-to-sink N partitioning with consequences on plant growth and development.

Identification and characterization of chloroplast amino acid transporters in Arabidopsis thaliana

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