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1.
J Exp Bot ; 55(406): 2155-68, 2004 Oct.
Article in English | MEDLINE | ID: mdl-15361541

ABSTRACT

Amino acids are regarded as the nitrogen 'currency' of plants. Amino acids can be taken up from the soil directly or synthesized from inorganic nitrogen, and then circulated in the plant via phloem and xylem. AtAAP3, a member of the Amino Acid Permease (AAP) family, is mainly expressed in root tissue, suggesting a potential role in the uptake and distribution of amino acids. To determine the spatial expression pattern of AAP3, promoter-reporter gene fusions were introduced into Arabidopsis. Histochemical analysis of AAP3 promoter-GUS expressing plants revealed that AAP3 is preferentially expressed in root phloem. Expression was also detected in stamens, in cotyledons, and in major veins of some mature leaves. GFP-AAP3 fusions and epitope-tagged AAP3 were used to confirm the tissue specificity and to determine the subcellular localization of AtAAP3. When overexpressed in yeast or plant protoplasts, the functional GFP-AAP3 fusion was localized in subcellular organelle-like structures, nuclear membrane, and plasma membrane. Epitope-tagged AAP3 confirmed its localization to the plasma membrane and nuclear membrane of the phloem, consistent with the promoter-GUS study. In addition, epitope-tagged AAP3 protein was localized in endodermal cells in root tips. The intracellular localization suggests trafficking or cycling of the transporter, similar to many metabolite transporters in yeast or mammals, for example, yeast amino acid permease GAP1. Despite the specific expression pattern, knock-out mutants did not show altered phenotypes under various conditions including N-starvation. Microarray analyses revealed that the expression profile of genes involved in amino acid metabolism did not change drastically, indicating potential compensation by other amino acid transporters.


Subject(s)
Amino Acid Transport Systems, Basic/genetics , Arabidopsis Proteins/genetics , Arabidopsis/physiology , Cell Membrane/physiology , Plant Roots/physiology , Amino Acid Sequence , Amino Acid Transport Systems, Basic/metabolism , Arabidopsis Proteins/metabolism , Base Sequence , DNA Primers , DNA, Bacterial/genetics , Gene Expression Profiling , Genes, Reporter , Molecular Sequence Data , Mutagenesis, Insertional , Polymerase Chain Reaction , Subcellular Fractions/physiology , TATA Box
2.
J Biol Chem ; 277(47): 45338-46, 2002 Nov 22.
Article in English | MEDLINE | ID: mdl-12244056

ABSTRACT

Arabidopsis amino acid transporters (AAPs) show individual temporal and spatial expression patterns. A new amino acid transporter, AAP8 was isolated by reverse transcription-PCR. Growth and transport assays in comparison to AAP1-5 characterize AAP8 and AAP6 as high affinity amino acid transport systems from Arabidopsis. Histochemical promoter-beta-glucuronidase (GUS) studies identified AAP6 expression in xylem parenchyma, cells requiring high affinity transport due to the low amino acid concentration in xylem sap. AAP6 may thus function in uptake of amino acids from xylem. Histochemical analysis of AAP8 revealed stage-dependent expression in siliques and developing seeds. Thus AAP8 is probably responsible for import of organic nitrogen into developing seeds. The only missing transporter of the family AAP7 was nonfunctional in yeast with respect to amino acid transport, and expression was not detectable. Therefore, AAP6 and -8 are the only members of the family able to transport aspartate with physiologically relevant affinity. AAP1, -6 and -8 are the closest AAP paralogs. Although AAP1 and AAP8 originate from a duplicated region on chromosome I, biochemical properties and expression pattern diverged. Overlapping substrate specificities paired with individual properties and expression patterns point to specific functions of each of the AAP genes in nitrogen distribution rather than to mere redundancy.


Subject(s)
Amino Acid Transport Systems/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Seeds/metabolism , Amino Acid Transport Systems/classification , Amino Acid Transport Systems/genetics , Arabidopsis/anatomy & histology , Arabidopsis/genetics , Arabidopsis Proteins/classification , Arabidopsis Proteins/genetics , Cloning, Molecular , Exons/genetics , Glucuronidase/genetics , Glucuronidase/metabolism , Introns/genetics , Plant Structures/metabolism , Promoter Regions, Genetic , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Nicotiana/anatomy & histology , Nicotiana/genetics , Nicotiana/metabolism , Yeasts/genetics , Yeasts/metabolism
3.
Plant J ; 29(6): 717-31, 2002 Mar.
Article in English | MEDLINE | ID: mdl-12148530

ABSTRACT

Amides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) from Arabidopsis mediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression in Xenopus oocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to specificity. Apparent substrate affinities are influenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating efficient transport of the basic amino acids. In vivo competition shows that the capability to transport basic amino acids in planta might be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an affinity for aspartate in the physiologically relevant range. This property is due to an overall higher affinity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower affinity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant deficient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transported in vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.


Subject(s)
Amino Acid Transport Systems/metabolism , Amino Acid Transport Systems/physiology , Algorithms , Amino Acid Transport Systems, Acidic/metabolism , Amino Acid Transport Systems, Acidic/physiology , Amino Acid Transport Systems, Basic/metabolism , Amino Acid Transport Systems, Basic/physiology , Amino Acid Transport Systems, Neutral/physiology , Amino Acids/metabolism , Animals , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Binding, Competitive , Female , Gene Expression Regulation, Enzymologic , Ion Transport , Membrane Potentials/physiology , Oocytes/metabolism , Patch-Clamp Techniques , Substrate Specificity , Xenopus laevis
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