Characterization of the rice glup4 mutant suggests a role for the small GTPase Rab5 in the biosynthesis of carbon and nitrogen storage reserves in developing endosperm

Abstract

Rice is unique among plants in that it accumulates major amounts of two types of seed storage proteins,prolamines and glutelins, which, along with starch, serve as sources of carbon and nitrogen for the post-germinative seedling. Here, we investigate the role of the small GTPase Rab5 in the biosynthesis of storageproteins through characterization of the loss-of-function mutant glup4. In situ RT-PCR and fluorescencemicroscopy revealed the glup4 mutant is impaired in glutelin mRNA localization to the cortical endoplasmicreticulum in developing endosperm cells. Furthermore, microarray and two-dimensional difference in gelelectrophoresis analysis of transcript and protein abundance, respectively, identified a number of genes whoseexpression is affected by the loss of Rab5, including starch biosynthetic enzymes. Our results indicate Rab5is important for RNA and protein sorting in rice seed and supports previous observations that the biosynthesisof starch and storage proteins may be interrelated.Key Words: rice, seed storage protein, RNA localization, seed storage reserves.IntroductionSeeds accumulate large amounts of storage reserves synthe-sized from translocated photoassimilates which are utilizedby the developing embryo and germinating seedling. Incereal seeds, these reserves are in the form of starch andstorage proteins and provide the main source of carbon andnitrogen, respectively. Although seemingly unrelated, evi-dence suggests that the biosyntheses of these storage prod-ucts may actually be coordinated (Giroux et al. 1996,Muller-Rober et al. 1992, Nelson 1982, Stark et al. 1992).As seeds are a vital component of the human diet throughoutmuch of the world, understanding the mechanism underly-ing this possible interrelatedness is of great importance.One approach to investigate this relationship is to identifyand characterize mutants defective in starch or storage pro-tein synthesis. Chemical mutagenesis by N-Methyl-N-Nitrosourea (MNU) of rice (Oryza sativa) has proven to bean effective method for inducing mutations at a high rate offrequency and has yielded a large collection of mutant pop-ulations (Suzuki et al. 2008), including those impaired instorage protein biosynthesis. Rice endosperm cells uniquelyaccumulate major quantities of two classes of storage pro-teins, prolamines, typical of cereals, and glutelins, homolo-gous to 11S globulins that are accumulated by legumes(Muench and Okita 1997). Protein synthesis occurs on theendoplasmic reticulum (ER), but the resulting polypeptidesare packaged into separate intracellular compartments: pro-lamine accumulates within the ER lumen, while glutelin issorted to protein storage vacuoles via the Golgi apparatus(Krishnan et al. 1986, Yamagata et al. 1982). Interestingly,proper protein sorting to these distinct compartments hasbeen found to be dependent upon the targeted transport oftheir RNAs to different subdomains of the cortical ER (re-viewed in Crofts et al. 2005). Prolamine mRNAs are local-ized to ER that delimits prolamine intracisternal inclusiongranules, while glutelin RNAs are directed to the adjacentcisternal ER (Choi et al. 2000). This targeted transport re-quires both cis- and trans-acting factors, although the exactmechanism remains unknown. The spatial separation ofmRNAs and subsequent protein synthesis may serve to