L-Glycine ● L-Serine ● L-Threonine ● L-Tyrosine ● L-Glutamine ● L-Glutamate ● L-Aspartate ● D-Aspartate ● L-Alanine ● L-Leucine ● L-Isoleucine ● L-Proline ● L-Methionine ● L-Lysine ● L-Arginine ● L-Tryptophan ● Uptake and intercellular transport of radio-labelled amino acids fed via the transpiration stream in Arabidopsis thaliana Daniel J. Kinsman, Ben G. Palmer, W. Paul Quick Department of Animal and Plant Sciences, University of Sheffield, UK Introduction: Nitrogen, a key resource for plants, is required for the synthesis of proteins and many other important types of molecule. Amino acids are the major ‘nitrogen currency’ of plants, being translocated between different cells and to different organs in response to the needs of the plant. This places great importance on the transporter proteins which carry amino acids across cellular membranes. In the genome of the model plant Arabidopsis thaliana, Over 50 putative amino acid transporters have been identified. The transporters characterised thus far frequently have broad, but different, substrate specificities and have affinities that range from the micromolar to the millimolar range (Fischer et al., 2002). Significantly, it appears that in many cases these genes show distinct expression patterns, suggesting spatial heterogeneity of the amino acid distribution system. Our aim was to characterise amino acid transport within the leaves of Arabidopsis. Leaves are important because they are the main recipients of xylem sap, which supplies the shoot with nitrogen, and are responsible for its redistribution to newly developing tissues. We have used dual-isotope imaging to provide spatial information on the uptake of amino acids fed simultaneously with sucrose to Arabidopsis thaliana leaves. Method: ● Individual mature leaves were excised from 5 week old short-day grown Arabidopsis plants. ● The radiolabel was introduced to the petiole to allow direct uptake into the xylem. The leaves were fed for 30 minutes and then transferred to a chase solution for 15 minutes. ● The solutions contained 0.1mM Sucrose and 0.1mM of one of the sixteen different amino acids that have been imaged to date. The feeding solution was spiked with 37kBq 14 C Sucrose and 1110kBq 3 H amino acid. ● At the end of the chase the leaves were rapidly frozen at -80 o C before freeze-drying to prepare them for imaging using a μ-imager autoradiography system (Biospace, France). ● The data for the two different isotopes were separated using the Energy Dual Label program (Biospace) and the resultant images normalised to the same scale relative to total counts imaged. Further work: ● Use of Radio-HPLC methods to analyse the metabolic fate of the label. ● Whole plant feeding experiments with 3 week old Arabidopsis plants. ● Feeding experiments with knock-out mutants of amino acid transporter genes. None of the lines thus far reported show an easily perceived phenotype, however, it is hoped that our method will reveal transport differences within the plants. Conclusions: The images clearly demonstrate that different amino acids have distinct sites of uptake within the leaf. For example, the basic amino acids Lysine and Arginine both show strong accumulation around the midrib and primary lateral veins, suggesting that high affinity uptake by cells in these areas results in very little label reaching the minor veins. In contrast, Aspartate and its D-isomer accumulated only in the region of the minor veins. Other amino acids show very different patterns, revealing specific amino acid uptake abilities of different parts of the vascular system found in leaves. This study is the first to investigate uptake and distribution of a wide range of amino acids from the transpiration stream, providing insight into the in vivo function of amino acid transporters and the utilization of the variety of amino acids which have been reported to occur in xylem sap (Pilot et al., 2004). It is also the first published work using dual imaging in plant metabolic tracer studies. In this experiment, the 14 C label provides a control for the variation between individual leaves, which may be caused by damage incurred during handling, micro-environment of the leaf within the feeding set-up or inherent differences between leaves. References: Fischer WN, Loo DD, Koch W, Ludewig U, Boorer KJ, Tegeder M, Rentsch D, Wright EM, Frommer WB (2002) Low and high affinity amino acid H+-cotransporters for cellular import of neutral and charged amino acids. Plant J. 29(6), 717-731 Pilot G, Stransky H, Bushey DF, Pratelli R, Ludewig U, Wingate VP, Frommer WB (2004) Overexpression of GLUTAMINE DUMPER1 leads to hypersecretion of glutamine from Hydathodes of Arabidopsis leaves. Plant Cell. 16, 1827-1840 Results: Each pair of images consists of the 3 H signal representing the amino acid (above) and the 14 C image representing the sucrose control (below). Key: ● Neutral amino acid ● Acidic amino acid ● Basic amino acid ● Non-polar amino acid mv - minor vein, plv - primary lateral vein, mr - midrib 5mm mr plv mv Daniel acknowledges Lablogic Systems Limited for sponsorship of his PhD and for assistance with the μ-imager and radio-HPLC work 3 H HPLC trace 14 C fed Arabidopsis plant
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