Prolamine and glutelin RNAs are localized to two subdomains of the cortical endoplasmic reticulum (ER), the protein body ER and the cisternal ER, in developing rice seeds. The addition of nearly full-length prolamine sequences at the 3 untranslated region of a reporter RNA redirects its localization from the cisternal ER to the protein body ER. Deletion analysis of prolamine RNA sequences indicates the presence of two partially redundant cis elements required for protein body ER targeting. The addition of glutelin 3 untranslated region to protein body ER cis sequences, however, redirects RNA localization to the cisternal ER. These results indicate that there are at least two regulated RNA transport pathways as well as a constitutive pathway to the cortical ER.

RNAs that code for the major rice storage proteins are localized to specific subdomains of the cortical endoplasmic reticulum (ER) in developing endosperm. Prolamine RNAs are localized to the ER and delimit the prolamine intracisternal inclusion granules (PB-ER), whereas glutelin RNAs are targeted to the cisternal ER. To study the transport of prolamine RNAs to the surface of the prolamine protein bodies in living endosperm cells, we adapted a two-gene system consisting of green fluorescent protein (GFP) fused to the viral RNA binding protein MS2 and a hybrid prolamine RNA containing tandem MS2 RNA binding sites. Using laser scanning confocal microscopy, we show that the GFP-labeled prolamine RNAs are transported as particles that move at an average speed of 0.3 to 0.4 m/s. These prolamine RNA transport particles generally move unidirectionally in a stop-and-go manner, although nonlinear bidirectional, restricted, and nearly random movement patterns also were observed. Transport is dependent on intact microfilaments, because particle movement is inhibited rapidly by the actin filament-disrupting drugs cytochalasin D and latrunculin B. Direct evidence was obtained that these prolamine RNA-containing particles are transported to the prolamine protein bodies. The significance of these results with regard to protein synthesis in plants is discussed.

SummaryPrevious studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of OsTudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. OsTudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that OsTudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that OsTudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of OsTudor-SN particles in transgenic rice plants expressing GFPtagged prolamine RNA transport particles showed co-localization of OsTudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged OsTudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of OsTudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that OsTudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[ 14 C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO 2 . Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.Cereal grains contribute a significant portion of worldwide starch production. Unlike other plant tissue, starch biosynthesis in the endosperm storage organ of cereal grains is unique in its dependence on two ADP-Glc pyrophosphorylase (AGPase) isoforms Thorbjørnsen et al., 1996;Sikka et al., 2001), a major cytosolic enzyme and a minor plastidial one, to generate ADP-glucose (ADPglc), the sugar nucleotide utilized by starch synthases in the amyloplast (Cakir et al., 2015). The majority of ADPglc in cereal endosperm is generated in the cytosol from AGPase (Tuncel and Okita, 2013) as well as by Suc synthase (Tuncel and Okita, 2013;Bahaji et al., 2014) and subsequently transported into amyloplasts by the BRITTLE-1 (BT1) protein located at the plastid envelope (Cao et al., 1995;Shannon et al., 1998).The Bt1 gene, first identified in maize (Zea mays; Mangelsdorf, 1926) and isolated by Sullivan et al. (1991), encodes a major amyloplast membrane protein ranging from 39 to 44 kD (Cao et al., 1995). The BT1 protein and its homologs belong to the mitochondrial carrier family (Sullivan et al., 1991;Haferkamp, 2007), which has a diverse range of substrates (Patron et al., 2004;Leroch et al., 2005;Kirchberger et al., 2008). The assignment of BT1 protein as the ADPglc transporter in cereal endosperms was first proposed by Sullivan et al. (1991), and then it was characterized based on the increased ADPglc levels and reduced ADPglc import rate * Address correspondence to okita@wsu.edu and hikaru.satoh.682@ m.kyushu-u.ac.jp.The authors responsible for distribution ...

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