The Proteomic Analysis of Maize Endosperm Protein Enriched by Phos-tagtm Reveals the Phosphorylation of Brittle-2 Subunit of ADP-Glc Pyrophosphorylase in Starch Biosynthesis Process

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.

Protein and Proteome Atlas for Plants under Stresses: New Highlights and Ways for Integrated Omics in Post-Genomics Era

“…Plant propagation and development are two important aspects that could largely determine the life cycle and economic values of different crops. The special issue studied different plants species, including an energy plant Jatropha curcas [18], a biofuel tree Pongamia [19], and three economic crops [20,21,22]. Liu et al used combined analyses of the phosphoproteomics, physiological characteristics, and ultrastructure studies to identify the responses of J. curcas seedlings under chilling, and revealed significantly changed phosphoproteins under chilling stress [18].…”

“…On the basis of this idea, Zhao et al conducted a phosphorproteomic analysis between two maize inbred lines showing different resistance to salt stress, and found that the enhancement of potassium and sodium transportation, carbon, and redox-related metabolism could increase the salt resistance in maize [19]. In this issue, Yu et al applied a newly developed Phos-tagTM technology to identify 21 phosphorylated peptides of AGPase [20].…”

Label-Free Comparative Proteomic Analysis Combined with Laser-Capture Microdissection Suggests Important Roles of Stress Responses in the Black Layer of Maize Kernels

“…Neighboring inside the BL is an endosperm tissue called BETL, and outside is a pericarp IEP. The proteome of whole maize seeds, embryos, and endosperm has been intensively investigated in recent decades [23,25,[38][39][40][41][42][43][44]. Due to the difficulty of separating tissues from kernels, the proteome for some targeted seed parts has seldom been the focus, except for BETL, whose functions have been implied to mediate embryo-endosperm interactions and play a role in plant defense by this method [45][46][47][48] To date, no attempt has been focused on BL, and very little is known about its developmental processes, as well as the physiological and molecular roles of this tissue in kernel development.…”

Molecular Functions and Pathways of Plastidial Starch Phosphorylase (PHO1) in Starch Metabolism: Current and Future Perspectives

“…These enzymes, each with various tissue-specific and developmental specific isoforms of starch synthases (SSs, EC 2.4.1.21), starch branching enzymes (SBEs, EC 2.4.1.18), and starch debranching enzymes (DBEs, 3.2.1.41; mainly isoamylases (ISAs)) are the major groups of enzymes involved in starch metabolism. ADP-Glc pyrophosphorylase (AGPase, EC 2.7.7.27) is the key enzyme in the first mandatory step of starch biosynthesis that produces the adenosine diphosphate glucose ADP-Glc from glucose-1-phosphate (Glc-1-P) and ATP [ 25 ]. ADP-Glc acts as a substrate to extend the α-1,4 linked glucans through SSs.…”