Biochemical and Functional Characterization of Plastidial ADP-glucose Transporter HvBT1 in Barley
Soliman, Atta S
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Starch is the main storage biopolymer in cereal plants. Several enzymes and carrier proteins are involved in the starch biosynthesis process. ADP-glucose pyrophosphorylase (AGPase) has been characterized as a key factor in this process, which catalyzes the conversion of glucose 1-phosphate into ADP-glucose in the cytosol of the endospermic cell. The freshly synthesized ADP-glucose must be transported into amyloplasts by the activity of ADP-glucose transporter. In the current research, we have characterized HvBT1 biochemically in E. coli system. HvBT1 shows high affinity to ADP-glucose as a transport substrate in counter-exchange with ADP with affinities of 614 and 334 µM, respectively. The cellular and subcellular localization of HvBT1 indicated its target the amyloplasts envelopes. The comparison between two barley cultivars; Harrington and Golden Promise shed some light on the impact of HvBT1 on starch accumulation. Higher expression of AGPase and HvBT1 (10 fold) provide an ideal combination for improving starch yield, where starch content was higher by 2.5% in Harrington. Unlike Harrington, the expression of soluble starch synthase encoded genes was higher in Golden Promise which accumulates less starch. This result provided evidence of the importance of HvBT1 in starch synthesis process along with AGPase. Down-regulation of HvBT1 also provided a cement evidence of its effect on the starch accumulation process, where the knock down lines showed 17% lower starch and altered starch composition. Also, as a result of decreasing starch, protein content increased in the transgenic grains by 4-5 % of its content in the wild type, while β-glucan was 37% lower than the wild type control. Down-regulation of HvBT1 led to decrease the grain yield by ~ 30% as a result of increase the grain size. Also, it seems to have pleotropic effects on other starch synthesis genes, where AGPLs was down-regulated while the plastidial SSU genes, AGPS1b and S2 were up-regulated. Soluble starch synthases SS2a and SS3a were down-regulated, while SS2b was up-regulated in the transgenic plants. The accumulated evidences indicated that HvBT1 is a key factor in starch biosynthesis process.