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Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins

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ABSTRACT

Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.

No MeSH data available.


Phosphorylation analysis of SN119 and ND5181.(A) Phosphoprotein analysis of SN119 and ND5181. (B) Phosphorylated peptides analysis of SN119 and ND5181. (C) Phosphorylation sites of SN119 and ND5181. (D) Distribution of serine, threonine and tyrosine phosphorylation sites. (E) Analysis of the amino acids surrounding the identified phosphorylated residues by Motif-X.
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f3: Phosphorylation analysis of SN119 and ND5181.(A) Phosphoprotein analysis of SN119 and ND5181. (B) Phosphorylated peptides analysis of SN119 and ND5181. (C) Phosphorylation sites of SN119 and ND5181. (D) Distribution of serine, threonine and tyrosine phosphorylation sites. (E) Analysis of the amino acids surrounding the identified phosphorylated residues by Motif-X.

Mentions: The starch granule-binding proteins extracted from the purified starch granules at 20 DPA in both cultivars, with three biological replicates, were subjected to phosphoproteome analysis. The phosphopeptides were enriched using TiO2 microcolumns, and then identified by LC-MS/MS. A total of 93 phosphopeptides, containing 91 phosphorylated sites and 61 phosphoproteins, were identified (Fig. 3A–C). In ND5181, 89 phosphopeptides, containing 91 phosphorylated sites and 60 phosphoproteins, were identified (Supplemental Table S5). A total of 68 phosphopeptides, with 70 phosphorylated sites and 48 phosphoproteins, were identified in SN119 (Supplemental Table S5). Among the 93 phosphorylated sites, 67 Ser, 20 Thr and 6 Tyr sites were phosphorylated (Fig. 3D). Motif-X showed that only Ser motifs ([sP]) were enriched in both cultivars, based on accurate parameters (P < 10−6; Fig. 3E). Only phosphorylation sites P ≥ 0.75 were used for the subsequent analyses. All of the mass spectrometry phosphoproteome data obtained in this study are shown in Supplemental Table S4 and were deposited in the Proteome X change Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository, with the dataset identifier PXD00064628.


Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins
Phosphorylation analysis of SN119 and ND5181.(A) Phosphoprotein analysis of SN119 and ND5181. (B) Phosphorylated peptides analysis of SN119 and ND5181. (C) Phosphorylation sites of SN119 and ND5181. (D) Distribution of serine, threonine and tyrosine phosphorylation sites. (E) Analysis of the amino acids surrounding the identified phosphorylated residues by Motif-X.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015113&req=5

f3: Phosphorylation analysis of SN119 and ND5181.(A) Phosphoprotein analysis of SN119 and ND5181. (B) Phosphorylated peptides analysis of SN119 and ND5181. (C) Phosphorylation sites of SN119 and ND5181. (D) Distribution of serine, threonine and tyrosine phosphorylation sites. (E) Analysis of the amino acids surrounding the identified phosphorylated residues by Motif-X.
Mentions: The starch granule-binding proteins extracted from the purified starch granules at 20 DPA in both cultivars, with three biological replicates, were subjected to phosphoproteome analysis. The phosphopeptides were enriched using TiO2 microcolumns, and then identified by LC-MS/MS. A total of 93 phosphopeptides, containing 91 phosphorylated sites and 61 phosphoproteins, were identified (Fig. 3A–C). In ND5181, 89 phosphopeptides, containing 91 phosphorylated sites and 60 phosphoproteins, were identified (Supplemental Table S5). A total of 68 phosphopeptides, with 70 phosphorylated sites and 48 phosphoproteins, were identified in SN119 (Supplemental Table S5). Among the 93 phosphorylated sites, 67 Ser, 20 Thr and 6 Tyr sites were phosphorylated (Fig. 3D). Motif-X showed that only Ser motifs ([sP]) were enriched in both cultivars, based on accurate parameters (P < 10−6; Fig. 3E). Only phosphorylation sites P ≥ 0.75 were used for the subsequent analyses. All of the mass spectrometry phosphoproteome data obtained in this study are shown in Supplemental Table S4 and were deposited in the Proteome X change Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository, with the dataset identifier PXD00064628.

View Article: PubMed Central - PubMed

ABSTRACT

Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.

No MeSH data available.