Three Diverse Granule Preparation Methods for Proteomic Analysis of Mature Rice (Oryza sativa L.) Starch Grain

Starch is the primary form of reserve carbohydrate storage in plants. Rice (Oryza sativa L.) is a monocot whose reserve starch is organized into compounded structures within the amyloplast, rather than a simple starch grain (SG). The mechanism governing the assembly of the compound SG from polyhedral granules in apposition, however, remains unknown. To further characterize the proteome associated with these compounded structures, three distinct methods of starch granule preparation (dispersion, microsieve, and flotation) were performed. Phase separation of peptides (aqueous trypsin-shaving and isopropanol solubilization of residual peptides) isolated starch granule-associated proteins (SGAPs) from the distal proteome of the amyloplast and the proximal 'amylome' (the amyloplastic proteome), respectively. The term 'distal proteome' refers to SGAPs loosely tethered to the amyloplast, ones that can be rapidly proteolyzed, while proximal SGAPs are those found closer to the remnant amyloplast membrane fragments, perhaps embedded therein-ones that need isopropanol solvent to be removed from the mature organelle surface. These two rice starch-associated peptide samples were analyzed using nano-liquid chromatography-tandem mass spectrometry (Nano-HPLC-MS/MS). Known and novel proteins, as well as septum-like structure (SLS) proteins, in the mature rice SG were found. Data mining and gene ontology software were used to categorize these putative plastoskeletal components as a variety of structural elements, including actins, tubulins, tubulin-like proteins, and cementitious elements such as reticulata related-like (RER) proteins, tegument proteins, and lectins. Delineating the plastoskeletal proteome begins by understanding how each starch granule isolation procedure affects observed cytoplasmic and plastid proteins. The three methods described herein show how the technique used to isolate SGs differentially impacts the subsequent proteomic analysis and results obtained. It can thus be concluded that future investigations must make judicious decisions regarding the methodology used in extracting proteomic information from the compound starch granules being assessed, since different methods are shown to yield contrasting results herein. Data are available via ProteomeXchange with identifier PXD032314.