Abstract
AGPase catalyzes a key rate-limiting step that converts ATP and Glc-1-p into ADP-glucose and diphosphate in maize starch biosynthesis. Previous studies suggest that AGPase is modulated by redox, thermal and allosteric regulation. However, the phosphorylation of AGPase is unclear in the kernel starch biosynthesis process. Phos-tagTM technology is a novel method using phos-tagTM agarose beads for separation, purification, and detection of phosphorylated proteins. Here we identified phos-tagTM agarose binding proteins from maize endosperm. Results showed a total of 1733 proteins identified from 10,678 distinct peptides. Interestingly, a total of 21 unique peptides for AGPase sub-unit Brittle-2 (Bt2) were identified. Bt2 was demonstrated by immunoblot when enriched maize endosperm protein with phos-tagTM agarose was in different pollination stages. In contrast, Bt2 would lose binding to phos-tagTM when samples were treated with alkaline phosphatase (ALP). Furthermore, Bt2 could be detected by Pro-Q diamond staining specifically for phosphorylated protein. We further identified the phosphorylation sites of Bt2 at Ser10, Thr451, and Thr462 by iTRAQ. In addition, dephosphorylation of Bt2 decreased the activity of AGPase in the native gel assay through ALP treatment. Taking together, these results strongly suggest that the phosphorylation of AGPase may be a new model to regulate AGPase activity in the starch biosynthesis process.
Highlights
Maize is one of the three crops with the highest production in the world [1]
Dephosphorylating of ADP-Glc pyrophosphorylase (AGPase) subunit Bt2 decreased its activity in the native gel assay. These results suggested that the phosphorylation of AGPase may be a new model for regulating AGPase activity in the starch biosynthesis process
Bt2-Ser10 was specific for maize, potato, and tomato in 10 selected plants (Figure 6D). These results show that three sites were phosphorylated for Bt2 protein in maize endosperm and that phosphorylation regulation of Bt2 may be complicated in the starch synthesis process
Summary
Maize is one of the three crops with the highest production in the world [1]. Maize starch, accounting for about 70% of maize kernels, is widely used as main energy source to support the majority of the world’s population, but is used as a feedstock for the production of industrial material and bioethanol [2]. Kernel starch is a crucial factor in determining the yield and quality of maize. It is a main goal of breeders to increase starch content and improve the quality of starch in maize. AGPase catalyzes the synthesis of ADP-glucose and diphosphate from ATP and Glc-1-p [4], and the product ADP-glucose serves as the activated glucosyl donor in α-1, 4-glucan synthesis [5,6]. This reaction is a key rate-limiting step in maize endosperm starch synthesis. AGPase widely exists in plant leaves and cereal endosperm in a heterotetrameric α2β2 format and is composed of two identical large subunits (Sh2) and two identical small subunits (Bt2) in maize endosperm [7,8]
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