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.

Protein Extraction Methods Shape Much of the Extracted Proteomes

“…This approach also enriches the LAP fraction in protein extracts, allowing for their improved separation and detection. Extensive proteomic studies of purified organelles, such as chloroplasts (Hall et al, 2011 ; Piro et al, 2015 ), nuclei (Sikorskaite et al, 2013 ), mitochondria (Lang et al, 2011 ; Salvato et al, 2014 ), and starch granules (Xing et al, 2016 ), have characterized a number of organelle proteins, and defined their localization information.…”

Proteome Analysis of Potato Starch Reveals the Presence of New Starch Metabolic Proteins as Well as Multiple Protease Inhibitors

“…On the other hand, amylopectin is synthesized by the concerted activities of soluble starch synthases (SSs), starch branching enzymes (BEs) and starch debranching enzymes (DBEs). To our knowledge, among the SS isoforms, only SS1, SS2 and SS3 were identified by proteomic analysis of maize starch as well as in starches from potato, rice, pea, barley and wheat ( Denyer et al, 1993 ; Peng et al, 2000 ; Borén et al, 2004 ; Grimaud et al, 2008 ; Stensballe et al, 2008 ; Xing et al, 2016 ). A series of evidence show that SS4 is involved in starch initiation although the precise mechanism is still under investigation ( Roldan et al, 2007 ; Szydlowski et al, 2009 ; Raynaud et al, 2016 ; Seung et al, 2016 ).…”

“…ISA1 and ISA2 participate in starch synthesis and interact to form hetero and homo complexes where the catalytic activity is carried by ISA1 ( Delatte et al, 2005 ). DBEs seem to be predominantly soluble but ISA2 and PU were recently identified in association with starch in rice ( Xing et al, 2016 ; Yu and Wang, 2016 ).…”

Optimization of a virus-induced gene silencing system and functional elucidation of MaBAM9b in postharvest banana fruits

“…I 2 -KI staining is a visual technique for quickly identifying starch contents in plant organs. This technique has been widely applied in rice [27], Zea mays [28], and Arabidopsis [29]. The result of I 2 -KI staining is in uenced by factors such as staining solution and time.…”