Proteome Profile of Starch Granules Purified from Rice (Oryza sativa) Endosperm

Abstract

Starch is the most important food energy source in cereals. Many of the known enzymes involved in starch biosynthesis are partially or entirely granule-associated in the endosperm. Studying the proteome of rice starch granules is critical for us to further understand the mechanisms underlying starch biosynthesis and packaging of starch granules in rice amyloplasts, consequently for the improvement of rice grain quality. In this article, we developed a protocol to purify starch granules from mature rice endosperm and verified the quality of purified starch granules by microscopy observations, I2 staining, and Western blot analyses. In addition, we found the phenol extraction method was superior to Tris-HCl buffer extraction method with respect to the efficiency in recovery of starch granule associated proteins. LC-MS/MS analysis showed identification of already known starch granule associated proteins with high confidence. Several proteins reported to be involved in starch synthesis in prior genetic studies in plants were also shown to be enriched with starch granules, either directly or indirectly, in our studies. In addition, our results suggested that a few additional candidate proteins may also be involved in starch synthesis. Furthermore, our results indicated that some starch synthesis pathway proteins are subject to protein acetylation modification. GO analysis and KEGG pathway enrichment analysis showed that the identified proteins were mainly located in plastids and involved in carbohydrate metabolism. This study substantially advances the understanding of the starch granule associated proteome in rice and post translational regulation of some starch granule associated proteins.

Fig 1. Work flow diagram showing the steps of the starch granule purification.

Mature rice endosperm were used as the starting materials for this experiment. The final products were the purified starch granules used for further experiments in this report.

Fig 2. Image of purified starch granules and the intermediate products of purification.

A: I₂ stain image of the purified starch granules. Microscope observation with 40 × amplification; B: Cross section image of rice endosperm viewed by SEM. 6k amplification; C: Large endosperm fragment image under SEM. 6k amplification; D: Endosperm fragment image under SEM. 6k amplification; E: Partially purified starch granule image under SEM, 6k amplification; F: The sediments of grounded endosperm image under SEM, 6k amplification; G-I: Purified starch granule image at different magnifications under SEM. G: 1 k amplification; H: 6k amplification; I: 24 k amplification.

Fig 3. The pattern of starch granule proteins and total proteins of rice endosperm on SDS PAGE.

The proteins extracted from rice endosperm and rice starch granules using phenol extraction method were separated by 12% SDS-PAGE and visualized by Coomassie blue stain. M: Marker; T: Total proteins; G: Starch granule proteins.

Fig 4. Western blot image of the starch granule proteome. Same amount of proteins (25 μg per lane) were loaded.

A: Western blot images with different antibodies. The anti-bodies used were for V-ATPase E, V-ATPase A, Anti-Rubisco, cFBPase (Agrisera, Sweden). T: Total proteins; G: Starch granule proteins. B: Western blot image of protein acetylation of endosperm and starch granule proteins. Antibodies for acetylated Lysine (ImmuneChem) were used for Western blots. The source of proteins is indicated on the top of the lane. M: Protein marker; T: Total proteins extracted from endosperm; G: Proteins extracted from starch granules.

Fig 5. Distribution of identified proteins based on their peptide counts.

A: Protein peptide count numbers vs numbers of proteins. The proteins identified with the same peptide counts were grouped together to obtain the protein numbers. X-axis: protein peptide counts; Y-axis: number of proteins. B: Peptide count percentage distribution of 40 proteins with highest peptide counts (excluding storage proteins) in the chloroplast/amyloplast (red) and other organelles (blue).

Fig 6. KEGG pathways enriched in the starch granule proteome.

KEGG pathway enrichment analysis of starch granule proteome. The value of -log10 (Fisher's test p value) is shown.

Fig 7. Proteins enriched in starch and sucrose metabolic pathways.

The enzymes marked with yellow and blue color are proteins enriched in rice starch granule proteome.