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

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.

Figure 1. Comparison of the expression patterns of three starch granule-binding proteins in SN119 and ND5181.

(A) Close-up views of expression levels for GBSS I, SS I and SS IIa protein spots on 2-DE gels. (B) Expression patterns and proportion of GBSS I, SS I and SS IIa proteins in five stages of seed development in SN119 and ND5181.

Figure 2. 2D-DIGE images of proteins in ND5181 and SN119.

(A) Labeled proteins were visualized for all fluorophores. (B) Cy2, red, mixture of equal amounts of all proteins as the internal standard. (C) Cy5, green, for the SN119 protein sample. (D) Cy3, blue, for the ND5181 protein sample.

Figure 3. 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.

Figure 4. Phosphorylation of starch synthase-related enzymes.

(A) Analysis of the amino acid sequence of SS IIa (phosphorylated residues are indicated). (B–E) Three-dimensional structures of SS IIa, SS I, SBE I, SBE IIa. The red balls represent the common phosphorylated proteins in both SN119 and ND5181, and the blue ball represents the special phosphorylated protein only existed in SN119.

Figure 5. Immunolocalization of GBSSI, SS I, SS IIa, SBE I and SBE IIa in immature seeds (15 DPA).

S, starch granules; PB, protein body; Triangular arrowheads indicate gold particles.

Figure 6. Quantitative real-time PCR (qRT-PCR) analysis of 6 key genes related to starch synthesis in developing seeds.

Figure 7. Schematic representation of amylose and amylopectin synthesis in the endosperm.

The color coding of each row displays the changes in the expression of starch synthesis-related genes at the transcriptional level. The line graphs show the changes in the expression of of starch granule-binding proteins at the protein level. Phosphoproteins are indicated by lowercase letter p. Complexes consisting of SS I/SS IIa and SBE II were formed after phosphorylation, and these complexes synthesized and modified the chains. For example, following synthesis of excessively lengthy A/B1-chains by SS I/SS IIa, SBE II cuts and transfers them to form the next B1-chain.