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.
Highlights
Starch is the main storage compound in grains of cereals
Its biosynthesis is catalyzed by a number of enzymes, including ADPglucose Pyrophosphorylase (AGPase) that converts glucose-1phosphate, using ATP, into ADP-glucose (ADP-Glc)
In vitro starch synthesis in amyloplasts isolated from Risø13, a lys5 mutant of barley generated by EMS mutagenesis, is suppressed as a result of defect in a major plastidial protein, which was identified as ADP-glucose transporter and designated as HvNST1 [7] and HvBT1 [8]
Summary
Starch is the main storage compound in grains of cereals. Its biosynthesis is catalyzed by a number of enzymes, including ADPglucose Pyrophosphorylase (AGPase) that converts glucose-1phosphate, using ATP, into ADP-glucose (ADP-Glc). The brittle (BT1) mutant is deficient in four amyloplasts envelope proteins, including ZmBT1, which is identified as an ADP-glucose transporter [3,4]. This mutant showed a lower rate of ADP-Glc uptake into isolated amyloplasts as compared to the control ones [5]. In vitro starch synthesis in amyloplasts isolated from Risø, a lys mutant of barley generated by EMS mutagenesis, is suppressed as a result of defect in a major plastidial protein, which was identified as ADP-glucose transporter and designated as HvNST1 [7] and HvBT1 [8]. Characterization of StBT1 using an E. coli expression system showed high affinity to AMP, ADP and ATP [9]
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