Biochemical and Molecular Characterization of Barley Plastidial ADP-Glucose Transporter (HvBT1)
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{"title"=>"Biochemical and molecular characterization of barley plastidial ADP-glucose transporter (HvBT1)", "type"=>"journal", "authors"=>[{"first_name"=>"Atta", "last_name"=>"Soliman", "scopus_author_id"=>"56205336200"}, {"first_name"=>"Belay T.", "last_name"=>"Ayele", "scopus_author_id"=>"15043517900"}, {"first_name"=>"Fouad", "last_name"=>"Daayf", "scopus_author_id"=>"6602711907"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "pui"=>"373323850", "doi"=>"10.1371/journal.pone.0098524", "sgr"=>"84902491543", "scopus"=>"2-s2.0-84902491543", "pmid"=>"24892865"}, "id"=>"594beb01-2ad9-3fa3-96ab-4d8e0d5e68bf", "abstract"=>"<p>In cereals, ADP-glucose transporter protein plays an important role in starch biosynthesis. It acts as a main gate for the transport of ADP-glucose, the main precursor for starch biosynthesis during grain filling, from the cytosol into the amyloplasts of endospermic cells. In this study, we have shed some light on the molecular and biochemical characteristics of barley plastidial ADP-glucose transporter, <italic>HvBT1</italic>. Phylogenetic analysis of several BT1 homologues revealed that BT1 homologues are divided into two distinct groups. The HvBT1 is assigned to the group that represents BT homologues from monocotyledonous species. Some members of this group mainly work as nucleotide sugar transporters. Southern blot analysis showed the presence of a single copy of <italic>HvBT1</italic> in barley genome. Gene expression analysis indicated that <italic>HvBT1</italic> is mainly expressed in endospermic cells during grain filling; however, low level of its expression was detected in the autotrophic tissues, suggesting the possible role of HvBT1 in autotrophic tissues. The cellular and subcellular localization of <italic>HvBT1</italic> provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of <italic>HvBT1</italic> using <italic>E. coli</italic> system revealed that HvBT1 is able to transport ADP-glucose into <italic>E. coli</italic> cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM. The study also showed that AMP is another possible exchange substrate. The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-<sup>32</sup>P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.</p>", "link"=>"http://www.mendeley.com/research/biochemical-molecular-characterization-barley-plastidial-adpglucose-transporter-hvbt1", "reader_count"=>5, "reader_count_by_academic_status"=>{"Researcher"=>1, "Student > Ph. D. Student"=>1, "Student > Master"=>2, "Unspecified"=>1}, "reader_count_by_user_role"=>{"Researcher"=>1, "Student > Ph. D. Student"=>1, "Student > Master"=>2, "Unspecified"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>2, "Unspecified"=>1}, "reader_count_by_subdiscipline"=>{"Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>1}}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1518931"], "description"=>"<p><i>Escherichia coli</i> C43 cells harboring the recombinant plasmid and the empty one as a control were incubated with different concentrations of [α-<sup>32</sup>P] ADP-Glc. The cells were incubated at 30°C for 10 min. The control values have been subtracted. The data are the mean ± SE of three independent experiments, each with three replicates. <b>A</b>: <i>K<sub>m</sub></i> value of ADP-glucose is 614.5±33.24 µM and <i>V<sub>max</sub></i> of 254.14 ±19.45 nmol of ADP-Glc mg of protein<sup>−1</sup> h<sup>−1</sup>. <b>B</b>: <i>K<sub>m</sub></i> and <i>V<sub>max</sub></i> values of ADP is 334.7±39.3 µM and of 47.07±3.51 nmol of ADP-Glc mg of protein<sup>−1</sup> h<sup>−1</sup>, respectively.</p>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "hvbt1"], "article_id"=>1044397, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524.g007", "stats"=>{"downloads"=>0, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Transport_activity_of_HvBT1_in_intact_E_coli_cells_/1044397", "title"=>"Transport activity of HvBT1 in intact <i>E. coli</i> cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-06-03 02:55:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1518930"], "description"=>"<p><i>Escherichia coli</i> C43 and rosetta2 strains harboring the original (org) or the optimized (opc) ORF of <i>HvBT1</i> were grown in 2xYT liquid media supplied with IPTG (0.5 mM final concentration). His-tagged HvBT1 membrane protein was purified and subjected to 12% SDS-PAGE. Lane 1 and 2 represent Rosetta 2 harboring opc and org ORF of <i>HvBT1</i>, respectively. Lane 3 and 4 represent C43 harboring opc and org ORF of <i>HvBT1</i>, respectively. Black arrows point to a band size of 45 KDa. Protein standard molecular weight is shown.</p>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "hvbt1"], "article_id"=>1044396, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524.g006", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_SDS_PAGE_analysis_of_HvBT1_protein_/1044396", "title"=>"SDS-PAGE analysis of HvBT1 protein.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-06-03 02:55:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1518927"], "description"=>"<p>Quantitative real-time RT-PCR was used to determine the expression level of <i>HvBT1</i> in different tissues using gene specific primers. <i>β-actin</i>, a housekeeping gene from barley, was used as a reference gene Seed samples during grain filling (2 to 20 DAA) and autotrophic tissue samples (stem and leaf) were used for gene expression analysis (see inset).</p>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "qpcr"], "article_id"=>1044393, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524.g003", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Real_time_qPCR_analysis_of_HvBT1_in_different_tissues_/1044393", "title"=>"Real-time qPCR analysis of <i>HvBT1</i> in different tissues.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-06-03 02:55:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1518925"], "description"=>"<p>The BLASTp <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098524#pone.0098524-Altschul1\" target=\"_blank\">[24]</a> program was used to retrieve the amino acid sequences of proteins related to BT1. The retrieved amino acid sequences were aligned with the ClustalX program <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098524#pone.0098524-Thompson1\" target=\"_blank\">[25]</a>. Phylogenetic estimates were created by the Molecular Evolutionary Genetic Analysis (MEGA 5.2) program package <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098524#pone.0098524-Tamura1\" target=\"_blank\">[26]</a>. The gaps were eliminated from the analysis in MEGA by using complete deletion setting. The phylogenetic tree was generated with the Maximum Parsimony (PARS), Neighbour joining (NJ; setting JTT model), and Maximum likelihood (ML) methods. MEGA 5.2 was also used for determining the best fit substitution model for ML analysis; thus for ML analysis the JTT+G model was applied and for all programs the bootstrap option was selected (1000 replicates) in order to obtain estimates for the confidence levels of the major nodes present within the phylogenetic trees. The phylogenetic tree divided BT1 homologues into two main groups, represent monocotyledonous, and both monocotyledonous and dicotyledonous species. HvBT1 was located in the first group within a distinct subgroup (orange color) with that of wheat (GenBank ID: ACX68637), which was characterized as ADP-glucose transporter <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098524#pone.0098524-Bowsher1\" target=\"_blank\">[2]</a>. Another subgroup (red color) represent BT proteins from monocot species including that of <i>maize</i> (GenBank ID: ACF78275) which is characterized as ADP-glucose transporter. The second group contained BT proteins from both monocotyledonous and dicotyledonous species. Dicotyledonous species were assigned in a distinct subgroup (brown color). They mainly function as nucleotide transporter, for example the potato (GenBank ID: ABA 81858) and <i>Arabidopsis</i> (GenBank ID: XP 004960816) BT proteins are characterized as an adenine nucleotide transporter <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098524#pone.0098524-Leroch1\" target=\"_blank\">[9]</a>, <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098524#pone.0098524-Kirchberger2\" target=\"_blank\">[31]</a>.</p>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "bt1", "amino"], "article_id"=>1044391, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524.g001", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Phylogenetic_analysis_of_BT1_amino_acid_sequences_/1044391", "title"=>"Phylogenetic analysis of BT1 amino acid sequences.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-06-03 02:55:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1518926"], "description"=>"<p>Barley nuclear DNA was digested with <b>1</b>: BamHI, <b>2</b>: SalI, <b>3</b>: XhoI and <b>4</b>: KpnI and subjected to southern blot analysis. DNA probe was prepared with 700 bp of <i>HvBT1</i> cDNA and used for hybridization. Molecular weight of the standard DNA ladder was indicated in the image.</p>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "blot", "barley"], "article_id"=>1044392, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524.g002", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Southern_blot_analysis_of_barley_HvBT1_/1044392", "title"=>"Southern blot analysis of barley <i>HvBT1</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-06-03 02:55:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1518933"], "description"=>"a<p>Control  =  [α-<sup>32</sup>P] ADP-glucose (100 µM).</p>b<p>Data are means ± SE, n = 3.</p>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "metabolites", "adp-glucose", "activities"], "article_id"=>1044399, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524.t001", "stats"=>{"downloads"=>2, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effects_of_different_metabolites_on_32_P_ADP_glucose_transport_activities_of_HvBT1_/1044399", "title"=>"Effects of different metabolites on [α-<sup>32</sup>P] ADP-glucose transport activities of HvBT1.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-06-03 02:55:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1518934"], "description"=>"<div><p>In cereals, ADP-glucose transporter protein plays an important role in starch biosynthesis. It acts as a main gate for the transport of ADP-glucose, the main precursor for starch biosynthesis during grain filling, from the cytosol into the amyloplasts of endospermic cells. In this study, we have shed some light on the molecular and biochemical characteristics of barley plastidial ADP-glucose transporter, <i>HvBT1</i>. Phylogenetic analysis of several BT1 homologues revealed that BT1 homologues are divided into two distinct groups. The HvBT1 is assigned to the group that represents BT homologues from monocotyledonous species. Some members of this group mainly work as nucleotide sugar transporters. Southern blot analysis showed the presence of a single copy of <i>HvBT1</i> in barley genome. Gene expression analysis indicated that <i>HvBT1</i> is mainly expressed in endospermic cells during grain filling; however, low level of its expression was detected in the autotrophic tissues, suggesting the possible role of HvBT1 in autotrophic tissues. The cellular and subcellular localization of <i>HvBT1</i> provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of <i>HvBT1</i> using <i>E. coli</i> system revealed that HvBT1 is able to transport ADP-glucose into <i>E. coli</i> cells with an affinity of 614.5 µM and in counter exchange of ADP with an affinity of 334.7 µM. The study also showed that AMP is another possible exchange substrate. The effect of non-labeled ADP-glucose and ADP on the uptake rate of [α-<sup>32</sup>P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.</p></div>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "molecular", "characterization", "barley", "plastidial", "adp-glucose", "transporter"], "article_id"=>1044400, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524", "stats"=>{"downloads"=>2, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Biochemical_and_Molecular_Characterization_of_Barley_Plastidial_ADP_Glucose_Transporter_HvBT1_/1044400", "title"=>"Biochemical and Molecular Characterization of Barley Plastidial ADP-Glucose Transporter (HvBT1)", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-06-03 02:55:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1518932"], "description"=>"<p><b>A</b>: <i>E. coli</i> cells harboring the vector containing <i>HvBT1</i> and the control vector were incubated with 1 µM [α-<sup>32</sup>P] ADP-Glc at 30°C for 5 min. The assay buffer was diluted with non-labeled ATP, ADP, AMP, and ADP-Glc for indicated time points. The cells were filtered and washed under vacuum, and then measured for radioactivity. The data presented here are the mean ± SE of three independent experiments, each with three replicates. <b>B</b>: the procedures for ADP efflux assay was performed as described for ADP-Glc in (A) with ADP and ADP-Glc dilutions. <b>C</b>: <i>E. coli</i> C43 cells harboring the vector containing <i>HvBT1</i> and the control vector were preloaded with nucleotides at a final concentration of 1 mM, and then the cells were incubated at 30°C for 5 min. The cells were centrifuged and re-suspended in potassium phosphate buffer (50 mM, pH 7.2) with [α-<sup>32</sup>P] ADP-Glc at concentration of 100 µM at 30°C for 8 min. The data presented are the mean ± SE of three independent experiments, each with three replicates.</p>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "intracellular", "radiolabeled"], "article_id"=>1044398, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524.g008", "stats"=>{"downloads"=>1, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Exchange_of_the_intracellular_radiolabeled_substrates_/1044398", "title"=>"Exchange of the intracellular radiolabeled substrates.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-06-03 02:55:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1518929"], "description"=>"<p>Subcellular localization of HvBT1 was visualized by Zeiss confocal Laser scanning microscope. <b>A</b>: transient expression of HvBT1::YFP in living protoplasts; chlorophyll autoflorescence (red color), YFP fluorescence (yellow color) and merged image (orange color). <b>B</b>: immunolocalization of HvBT1::YFP was detected using anti-YFP antibody and visualized by the fluorescence of FITC-conjugated antibody. Images represent FITC fluorescence (blue color), bright field (grey image) and the merged image that show the localization of HvBT1::YFP on the chloroplasts membranes.</p>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "localization"], "article_id"=>1044395, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524.g005", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Subcellular_localization_of_HvBT1_YFP_/1044395", "title"=>"Subcellular localization of HvBT1::YFP.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-06-03 02:55:42"}
  • {"files"=>["https://ndownloader.figshare.com/files/1518928"], "description"=>"<p>Cellular localization of <i>HvBT1</i> was assayed using RNA-in situ hybridization. <b>A</b>: hybridization with the sense probe which produces a very faint signal. <b>B</b>: hybridization with antisense probe which produces high signal of alkaline phosphatase. <b>es</b>; embryo sac, <b>al</b>; aleurone, and <b>em</b>; embryo. Strong signal was detected in the embryo sac, which accumulates the endosperm.</p>", "links"=>[], "tags"=>["agriculture", "Agricultural biotechnology", "Genetically modified organisms", "Transgenic plants", "Plant products", "Biochemistry", "biosynthesis", "Carbohydrate biosynthesis", "Biochemical activity", "Plant biochemistry", "localization"], "article_id"=>1044394, "categories"=>["Biological Sciences"], "users"=>["Atta Soliman", "Belay T. Ayele", "Fouad Daayf"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0098524.g004", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cellular_localization_of_HvBT1_/1044394", "title"=>"Cellular localization of <i>HvBT1</i>.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-06-03 02:55:42"}

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