In developing rice () endosperm, mRNAs of the major storage proteins, glutelin and prolamine, are transported and anchored to distinct subdomains of the cortical endoplasmic reticulum. RNA binding protein RBP-P binds to both glutelin and prolamine mRNAs, suggesting a role in some aspect of their RNA metabolism. Here, we show that rice lines expressing mutant RBP-P mislocalize both glutelin and prolamine mRNAs. Different mutant RBP-P proteins exhibited varying degrees of reduced RNA binding and/or protein-protein interaction properties, which may account for the mislocalization of storage protein RNAs. In addition, partial loss of RBP-P function conferred a broad phenotypic variation ranging from dwarfism, chlorophyll deficiency, and sterility to late flowering and low spikelet fertility. Transcriptome analysis highlighted the essential role of RBP-P in regulating storage protein genes and several essential biological processes during grain development. Overall, our data demonstrate the significant roles of RBP-P in glutelin and prolamine mRNA localization and in the regulation of genes important for plant growth and development through its RNA binding activity and cooperative regulation with interacting proteins.

SUMMARYStudies focusing on the targeting of RNAs that encode rice storage proteins, prolamines and glutelins to specific sub-domains of the endoplasmic reticulum (ER), as well as mis-localization studies of other storage protein RNAs, indicate a close relationship between the ER site of RNA translation and the final site of protein deposition in the endomembrane system in developing rice endosperm. In addition to prolamine and glutelin, rice accumulates smaller amounts of a-globulins, which are deposited together with glutelin in the protein storage vacuole (PSV). In situ RT-PCR analysis revealed that a-globulin RNAs are not distributed to the cisternal ER as expected for a PSV-localized protein, but instead are targeted to the protein body-ER (PB-ER) by a regulated process requiring cis-sorting sequences. Sequence alignments with putative maize d-zein cis-localization elements identified several candidate regulatory sequences that may be responsible for PB-ER targeting. Immunocytochemical analysis confirmed the presence of a-globulin on the periphery of the prolamine protein bodies and packaging in Golgi-associated dense vesicles, as well as deposition and storage within peripheral regions of the PSV. Mis-targeting of a-globulin RNAs to the cisternal ER dramatically alters the spatial arrangement of a-globulin and glutelin within the PSV, with the accompanying presence of numerous small a-globulin particles in the cytoplasm. These results indicate that a-globulin RNA targeting to the PB-ER sub-domain is essential for efficient transport of a-globulins to the PSV and its spatial arrangement in the PSV. Such RNA localization prevents potential deleterious protein-protein interactions, in addition to performing a role in protein targeting.

Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

In eukaryotes, RNA binding proteins (RBPs) play an integral role not only in RNA processing within the nucleus, but also in the cytoplasmic events of RNA transport, localization, translation, storage and degradation. While many studies have been done, relatively little is known about RBPs in plants. As part of our continuing efforts to understand cytoplasmic gene expression events in developing rice seed (Oryza sativa L.), a proteomics approach was used to identify cytoplasmic-localized, cytoskeletal-associated RBPs. The nucleic acid binding fraction from a cytoskeletal-enriched rice seed extract was isolated by Poly(U)-Sepharose affinity chromatography and analyzed using 2D gel electrophoresis. Analysis of 162 excised protein spots using mass spectrometry led to the identification of 148 distinct proteins, in addition to the highly abundant globulin and glutelin seed storage proteins. Identified proteins include those involved in RNA processing, translation, protein modification, cell signaling, and metabolism, as well as a number of hypothetical proteins. Proteins of particular interest with roles in RNA metabolism are discussed. These results have been deposited within the Rice RNA Binding Protein Database as part of an integrated study of plant cytoskeletal-associated RBPs using developing rice seed as a model.