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Biochemical and molecular characterization of barley plastidial ADP-glucose transporter (HvBT1).

Soliman A, Ayele BT, Daayf F - PLoS ONE (2014)

Bottom Line: Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli 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-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Genetics, Faculty of Agriculture, University of Tanta, Tanta, El-Gharbia, Egypt.

ABSTRACT
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, HvBT1. 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 HvBT1 in barley genome. Gene expression analysis indicated that HvBT1 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 HvBT1 provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli 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-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

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Phylogenetic analysis of BT1 amino acid sequences.The BLASTp [24] 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 [25]. Phylogenetic estimates were created by the Molecular Evolutionary Genetic Analysis (MEGA 5.2) program package [26]. 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 [2]. Another subgroup (red color) represent BT proteins from monocot species including that of maize (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 Arabidopsis (GenBank ID: XP 004960816) BT proteins are characterized as an adenine nucleotide transporter [9], [31].
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pone-0098524-g001: Phylogenetic analysis of BT1 amino acid sequences.The BLASTp [24] 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 [25]. Phylogenetic estimates were created by the Molecular Evolutionary Genetic Analysis (MEGA 5.2) program package [26]. 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 [2]. Another subgroup (red color) represent BT proteins from monocot species including that of maize (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 Arabidopsis (GenBank ID: XP 004960816) BT proteins are characterized as an adenine nucleotide transporter [9], [31].

Mentions: The phylogenetic analysis divided BT1 proteins into two main groups. The first group represented monocotyledonous species including wheat, barley, maize and rice, and the second group represented both monocotyledonous and dicotyledonous species (Figure 1). The first group consists of subgroups of BT1 homologues that are classified mainly based on their biochemical functions. The HvBT1 (GenBank ID: AAT12275) is classified in a distinct subgroup (orange color) that includes BT1 homologues from Triticum aestivum and Triticum urartu. Comparison of the amino acid sequence of HvBT1 with that of other BT homologues showed high similarity with BT homologue from Triticum aestivum (GenBank ID: ACX68637; with 97% similarity or 92% identity), Triticum urartu (GenBank ID: EMS62502; with 99% similarity or 88% identity), Aegilopus tauschii (GenBank ID: EMT17313; with 99% similarity or 89% identity) and Aegilopus crassa (GenBank ID: ACX68638; with 97% similarity or 90% identity). The HvBT1 also showed similarity with homologues from Zea mays (GenBank ID: NP001105889; with 92% similarity or 66% identity) and Zea mays (GenBank ID: ACF78275; with 92% similarity or 71% identity). Other subgroups include BT1 homologues that function as either nucleotide sugar transporter or adenine nucleotide transporter. The second group consists of BT1 homologues from both monocotyledonous and dicotyledonous species. This group is divided into two main distinct subgroups. One of the subgroups includes BT1 from monocotyledonous species that mainly function as either nucleotide sugar transporter or adenine nucleotide transporter. The second subgroup includes BT1 homologues from only dicotyledonous species that mainly function as nucleotide carrier proteins. The BT1 homologues from dicotyledonous species showed low similarity to HvBT1.


Biochemical and molecular characterization of barley plastidial ADP-glucose transporter (HvBT1).

Soliman A, Ayele BT, Daayf F - PLoS ONE (2014)

Phylogenetic analysis of BT1 amino acid sequences.The BLASTp [24] 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 [25]. Phylogenetic estimates were created by the Molecular Evolutionary Genetic Analysis (MEGA 5.2) program package [26]. 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 [2]. Another subgroup (red color) represent BT proteins from monocot species including that of maize (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 Arabidopsis (GenBank ID: XP 004960816) BT proteins are characterized as an adenine nucleotide transporter [9], [31].
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pone-0098524-g001: Phylogenetic analysis of BT1 amino acid sequences.The BLASTp [24] 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 [25]. Phylogenetic estimates were created by the Molecular Evolutionary Genetic Analysis (MEGA 5.2) program package [26]. 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 [2]. Another subgroup (red color) represent BT proteins from monocot species including that of maize (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 Arabidopsis (GenBank ID: XP 004960816) BT proteins are characterized as an adenine nucleotide transporter [9], [31].
Mentions: The phylogenetic analysis divided BT1 proteins into two main groups. The first group represented monocotyledonous species including wheat, barley, maize and rice, and the second group represented both monocotyledonous and dicotyledonous species (Figure 1). The first group consists of subgroups of BT1 homologues that are classified mainly based on their biochemical functions. The HvBT1 (GenBank ID: AAT12275) is classified in a distinct subgroup (orange color) that includes BT1 homologues from Triticum aestivum and Triticum urartu. Comparison of the amino acid sequence of HvBT1 with that of other BT homologues showed high similarity with BT homologue from Triticum aestivum (GenBank ID: ACX68637; with 97% similarity or 92% identity), Triticum urartu (GenBank ID: EMS62502; with 99% similarity or 88% identity), Aegilopus tauschii (GenBank ID: EMT17313; with 99% similarity or 89% identity) and Aegilopus crassa (GenBank ID: ACX68638; with 97% similarity or 90% identity). The HvBT1 also showed similarity with homologues from Zea mays (GenBank ID: NP001105889; with 92% similarity or 66% identity) and Zea mays (GenBank ID: ACF78275; with 92% similarity or 71% identity). Other subgroups include BT1 homologues that function as either nucleotide sugar transporter or adenine nucleotide transporter. The second group consists of BT1 homologues from both monocotyledonous and dicotyledonous species. This group is divided into two main distinct subgroups. One of the subgroups includes BT1 from monocotyledonous species that mainly function as either nucleotide sugar transporter or adenine nucleotide transporter. The second subgroup includes BT1 homologues from only dicotyledonous species that mainly function as nucleotide carrier proteins. The BT1 homologues from dicotyledonous species showed low similarity to HvBT1.

Bottom Line: Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli 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-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Science, Faculty of Agricultural and Food Sciences, University of Manitoba, Winnipeg, Manitoba, Canada; Department of Genetics, Faculty of Agriculture, University of Tanta, Tanta, El-Gharbia, Egypt.

ABSTRACT
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, HvBT1. 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 HvBT1 in barley genome. Gene expression analysis indicated that HvBT1 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 HvBT1 provided additional evidence that HvBT1 targets the amyloplast membrane of the endospermic cells. Biochemical characterization of HvBT1 using E. coli system revealed that HvBT1 is able to transport ADP-glucose into E. coli 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-32P] ADP-glucose indicated the substrate specificity of HvBT1 for ADP-glucose and ADP.

Show MeSH