Control of Storage Protein Biosynthesis by mRNA Targeting

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The major metabolic activities of plant seeds are directed toward the synthesis of carbon (in the form of lipids and starch) and nitrogen (in the form of storage proteins) reserves. Although the biochemical processes responsible for the biosynthesis of these macromolecules appear to be independent processes, evidence gathered over the years have indicated otherwise. Carbon and nitrogen metabolism are inter-related as evidenced by the almost parallel responses exhibited by defined genetic mutants and transgenic plants altered in gene expression in one of these two processes. The mechanisms responsible for this coordinated expression of carbon and nitrogen reserves are not known. This grant proposes a series of studies to study one aspect of nitrogen metabolism, i.e. the control of storage protein biosynthesis in developing rice endosperm. Rice is an excellent system to study storage protein biosynthesis (and its relationship to starch synthesis) in that it is one of the few plants that synthesize and accumulate both major classes of storage proteins, prolamines and glutelins, the latter an 11S globulin. Moreover, rice stores these proteins in different subcellular compartments. Prolamines are deposited directly as protein intracisternal granules within the endoplasmic reticulum (ER) lumen, whereas glutelins are transported through the ER and packaged in a protein storage vacuole. Previous studies from this laboratory have demonstrated that the RNAs encoding these two classes of storage proteins are not localized in a stochastic fashion on the ER. Instead, prolamine mRNAs are highly enriched on the ER membranes that bound the prolamine protein bodies (PBs) whereas glutelin mRNAs predominant on the cisternal ER. The majority of the prolamine PBs is distributed near the plasma membrane in the cortex, a region enriched in cytoskeleton. The localization of prolamine mRNAs to the cortical region, a distance of 7-15 um from the nucleus, suggests that the prolamine RNA itself, and not the coded protein, is targeted to the prolamine PBs. Indeed, results from ongoing studies on the localization of synthetic prolamine RNA transcripts in transgenic rice indicate that these RNAs are targeted to the prolamine PBs because they contain specific "zip code" signals. To better understand this process, experiments are described that will attempt to identify these "zip codes", and to identify and characterize the zip code binding proteins, which are responsible for the transport and anchoring of the prolamine RNAs to the prolamine PBs. Efforts will also be made to reconstruct prolamine RNA movement in cultured and developing endosperm cells, which will provide cellular details on the intracellular highways for RNA movement and provide clues on the nature of the molecular motor responsible for RNA movement. A second general goal of the project is to determine whether glutelin RNAs also possess "zip codes" that target it to a different ER subdomain and to determine the spatial relationship between prolamine and glutelin RNA localization in the cell. Future studies will address the intracellular location of RNAs that code for enzymes involved in starch biosynthesis. An integrated approach will be taken to achieve these goals, and will incorporate the use of biochemical, cellular, molecular and ultrastructural techniques. This study will provide new information on how RNAs and, in turn, proteins are sorted in plant cells and how the ER can differentiate into unique domains with specific functions. Moreover, it will lead to new insights into the mechanisms that may control utilization of carbon and nitrogen and their conversion into storage reserves.