Rice (Oryza sativa) accumulates prolamins and glutelins as storage proteins. The latter storage protein is synthesized on the endoplasmic reticulum (ER) as a 57-kD proglutelin precursor, which is then processed into acidic and basic subunits in the protein storage vacuole. Three esp2 mutants, CM1787, EM44, and EM747, contain larger amounts of the 57-kD polypeptide and corresponding lower levels of acidic and basic glutelin subunits than normal. Electron microscopic observation revealed that esp2 contained normal-appearing glutelin-containing protein bodies (PB-II), but lacked the normal prolamin-containing PB (PB-I). Instead, numerous small ER-derived PBs of uniform size (0.5 m in diameter) and low electron density were readily observed. Immunoblot analysis of purified subcellular fractions and immunocytochemistry at the electron microscopy level showed that these new PBs contained the 57-kD proglutelin precursor and prolamin polypeptides. The 57-kD proglutelin was extracted with 1% (v/v) lactic acid solution only after removal of cysteine-rich prolamin polypeptides, suggesting that these proteins form glutelin-prolamin aggregates via interchain disulfide bonds within the ER lumen. The endosperm of esp2 mutants contains the lumenal chaperones, binding protein and calnexin, but lacks protein disulfide isomerase (PDI) at the protein and RNA levels. The transcript of PDI was expressed in the seed only during the early stage of seed development in the wild type. These results suggest that PDI plays an essential role in the segregation of proglutelin and prolamin polypeptides within the ER lumen.All plants utilize storage proteins as a reserve of nitrogen, sulfur, and carbon in the form of salt-soluble globulins or alcohol-soluble prolamins (Shewry and Casey, 1999). In addition to the alcohol-soluble prolamins typically found in cereals (Shewry and Tatham, 1999), rice (Oryza sativa) also accumulates glutelins, which are proteins homologous to the 11S globulin of soybean (Glycine max) and pea (Pisum sativum; Zhao et al., 1983;Takaiwa et al., 1987;Shotwell and Larkins, 1989). Although both storage proteins are initially synthesized on the endoplasmic reticulum (ER) membrane (Yamagata et al., 1982) and are translocated into the ER lumen, they are stored in morphologically distinct protein bodies (PB; Tanaka et al., 1980; Krishnan et al., 1986;Yamagata and Tanaka, 1986). Prolamins are stored as intracisternal inclusion granules within the ER lumen (PB-I), whereas glutelins are packaged in a protein storage vacuolar compartment (PB-II; Tanaka et al., 1980; Krishnan et al., 1986). These PBs are readily distinguishable at the light and electron microscopy levels. The prolamincontaining PB-I is spherical, with a diameter of about 1 to 2 m, and exhibits concentric rings of varying electron density (Bechtel and Juliano, 1980;Tanaka et al., 1980; Krishnan et al., 1986;Yamagata and Tanaka, 1986). In contrast, the glutelin-containing PB-II is larger (3-4 m), irregularly shaped, and of highly uniform staining density.The cellular...

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Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.

Acid-soluble collagen (ASC) and pepsin solubilized collagen (PSC) were isolated from the skins of black drum (Pogonias cromis) and sheepshead seabream (Archosargus probatocephalus) harvested in the Gulf of Mexico coastal waters. The yields of ASCs on dry basis from black drum and sheepshead were estimated at 2.3 and 2.6%, and the yields of PSCs were 15.8 and 29.3%, respectively. Analyses of molecular weight profile, amino acid composition, and secondary structure showed that the skin collagens from both species were typical type-I collagen. The molecular mass of alpha(1) and alpha(2) subunits, as determined by SDS-PAGE using Tris-Acetate gels, was 127 kDa and 116 kDa, respectively. The amino acid composition of ASC and PSC for both species was closer to calf skin ASC than to cod skin ASC. Thermal denaturation temperatures, measured by melting point using circular dichroism, gave the following values: black drum ASC, 34.2 degrees C; sheepshead ASC, 34.0 degrees C; black drum PSC, 35.8 degrees C; sheepshead PSC, 34.3 degrees C. The literature value for the heat stability of calf skin collagen is 36.3 degrees C. The potentials of collagens from black drum and sheepshead skins in the functional food, healthcare, and pharmaceutical industries are discussed.

In the rice (Oryza sativa) endosperm, storage proteins are synthesized on the rough endoplasmic reticulum (ER), in which prolamins are sorted to protein bodies (PBs) called type-I PB (PB-I). Protein disulfide isomerase (PDI) family oxidoreductase PDIL2;3, an ortholog of human P5, contains a conserved structural disulfide in the redox-inactive thioredoxin-like (TRX) domain and was efficiently targeted to the surface of PB-I in a redox active site-dependent manner, whereas PDIL1;1, an ortholog of human PDI, was localized in the ER lumen. Complementation analyses using PDIL1;1 knockout esp2 mutant indicated that the a and a9 TRX domains of PDIL1;1 exhibited similar redox activities and that PDIL2;3 was unable to perform the PDIL1;1 functions. PDIL2;3 knockdown inhibited the accumulation of Cys-rich 10-kD prolamin (crP10) in the core of PB-I. Conversely, crP10 knockdown dispersed PDIL2;3 into the ER lumen. Glutathione S-transferase-PDIL2;3 formed a stable tetramer when it was expressed in Escherichia coli, and the recombinant PDIL2;3 tetramer facilitated a-globulin(C79F) mutant protein to form nonnative intermolecular disulfide bonds in vitro. These results indicate that PDIL2;3 and PDIL1;1 are not functionally redundant in sulfhydryl oxidations of structurally diverse storage proteins and play distinct roles in PB development. We discuss PDIL2;3-dependent and PDIL2;3-independent oxidation pathways that sustain disulfide bonds of crP10 in PB-I.

Saline-soluble glycinins and insoluble glutelins are the major storage proteins in soybean (Glycine max) and rice (Oryza sativa), respectively. In spite of their differences in solubility properties, both proteins are members of the 11S globulin gene family based on their similarities in primary sequences and processing of the coded protein. Wild-type and methionine-modified glycinin coding sequences were expressed in transgenic rice plants under the control of the rice glutelin GluB-1 promoter. Glycinins were specifically synthesized in the endosperm tissue and co-localized with glutelins in type II protein bodies. They assembled into 7S and 11S species, similar to what was observed in developing soybean seeds. This pattern was quite different from that displayed by the rice glutelins in untransformed plants, in which processed subunits sedimenting at 2S were apparent. In glycinin-expressing transgenic plants, however, glutelins were observed sedimenting at 7S and 11S with lesser amounts in the 2S region. A portion of the glycinins was also found associated in the insoluble glutelin fraction. Renaturation experiments suggested that the hybrid glycinin-glutelin oligomers were formed through specific interactions. Overall, these results indicate that despite significant differences in the assembly of soybean glycinin and rice glutelin, both proteins can assemble with each other to form soluble hexameric oligomers or insoluble aggregates.Seed storage proteins were initially classified into albumins (water soluble), globulins (saline soluble), prolamins (alcohol soluble), and glutelins (residue) by Osborne (1924) according to their solubility properties. Based on more recent and extensive molecular and biochemical analysis of the storage protein genes and their coded products, the storage proteins fall into two major groups, the globulins and the prolamins (Shewry and Tatham, 1990). The rice (Oryza sativa) glutelins and soybean (Glycine max) glycinins are excellent examples of this reclassification of storage proteins. The rice glutelins, which comprise up to 70% to 80% of the total seed protein, are insoluble in a neutral saline solution but soluble in a diluted acid/alkaline solution. They exist as large macromolecular complexes formed by disulfide and hydrophobic interactions of acidic and basic polypeptides. The soybean glycinins, which account for 40% of the total proteins (Utsumi, 1992; Utsumi et al., 1997), are soluble in neutral saline solutions. These proteins accumulate as 11S oligomers comprised of six pairs (subunits) of acidic and basic polypeptides interlinked by a conserved disulfide bond. Although glutelin and glycinin have different properties (such as their solubility), they nevertheless are related and are both members of the 11S globulin family of storage proteins. These proteins share 32% to 37% identity in their primary sequences. Moreover, both proteins are synthesized as a larger precursor on the ER, are proteolytically processed into acidic and basic polypeptides, and are accumulated and...