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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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Southern blot analysis of barley HvBT1.Barley nuclear DNA was digested with 1: BamHI, 2: SalI, 3: XhoI and 4: KpnI and subjected to southern blot analysis. DNA probe was prepared with 700 bp of HvBT1 cDNA and used for hybridization. Molecular weight of the standard DNA ladder was indicated in the image.
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pone-0098524-g002: Southern blot analysis of barley HvBT1.Barley nuclear DNA was digested with 1: BamHI, 2: SalI, 3: XhoI and 4: KpnI and subjected to southern blot analysis. DNA probe was prepared with 700 bp of HvBT1 cDNA and used for hybridization. Molecular weight of the standard DNA ladder was indicated in the image.

Mentions: Restriction analysis of barley genomic DNA with XhoI, BamHI, SalI and kpnI (Figure 2) revealed that only one distinct band can be produced by digestion with different restriction enzymes that do not cut the HvBT1 cDNA sequence. This result indicates that there is only one copy of HvBT1 in barley genome.


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

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

Southern blot analysis of barley HvBT1.Barley nuclear DNA was digested with 1: BamHI, 2: SalI, 3: XhoI and 4: KpnI and subjected to southern blot analysis. DNA probe was prepared with 700 bp of HvBT1 cDNA and used for hybridization. Molecular weight of the standard DNA ladder was indicated in the image.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043945&req=5

pone-0098524-g002: Southern blot analysis of barley HvBT1.Barley nuclear DNA was digested with 1: BamHI, 2: SalI, 3: XhoI and 4: KpnI and subjected to southern blot analysis. DNA probe was prepared with 700 bp of HvBT1 cDNA and used for hybridization. Molecular weight of the standard DNA ladder was indicated in the image.
Mentions: Restriction analysis of barley genomic DNA with XhoI, BamHI, SalI and kpnI (Figure 2) revealed that only one distinct band can be produced by digestion with different restriction enzymes that do not cut the HvBT1 cDNA sequence. This result indicates that there is only one copy of HvBT1 in barley genome.

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