Isolation and Characterization of Two Types of Protein Bodies in the Rice Endosperm

Two types of proteinaceous particles were observed under the electron microscope in the starchy endosperm of rice seeds. One was spherical with lamellar structure (PB-I), while the other was stained homogeneously by osmium tetroxide and not lamellar structured (PB-II). Both types of proteinaceous particles were effectively condensed from the homogenate of developing rice endosperm by an aqueous polymer two-phase system using dextran-DEAE dextran-polyethylene glycol. Separation of both types was carried out by sucrose density gradient centrifugation. These proteinaceous particles were recovered at specific gravities of 1.27 and 1.29 for PB-I and PB-II, respectively. The protein composition of these particles and their solubility fractionation were examined. Prolamin appeared in the PB-I fraction, whereas PB-II was rich in glutelin and globulin.     

Molecular Cloning, Expression and Subcellular Localization of a BiP Homolog from Rice Endosperm Tissue

The ER luminal binding protein, BiP, has been linked to prolamineprotein body formation in rice. To obtain further informationon the possible role of this chaperone in protein body formationwe have cloned and sequenced a BiP cDNA homolog from rice endosperm.The rice sequence is very similar to the maize BiP exhibiting92% nu-cleotide identity and 96% deduced amino acid sequenceidentity in the coding region. Substantial amino acid sequencehomology exists between rice BiP and BiP homo-logs from severalother plant and animal species including long stretches of conservationthrough the amino-terminal ATPase domain. Considerable variation,however, is observed within the putative carboxy-terminal peptide-bind-ingdomain between the plant and nonplant BiP sequences. A singleband of approximately 2.4 kb was visible when RNA gel blotsof total RNA purified from seed tissue were probed with radiolabeledrice BiP cDNA. This band increased in intensity during seeddevelopment up to 10 days after flowering, and then decreasedgradually until seed maturity. Protein gel blots indicated thatBiP polypeptide accumulation parallels that of the prolaminepolypeptides throughout seed development. Immunocytochemicalanalysis demonstrated that BiP is localized in a non-stochasticfashion in the endoplasmic reticulum membrane complex of developingendosperm cells. It is abundant on the periphery of the proteininclusion body but not in the central portion of the proteinbody or in the cisternal ER membranes connecting the proteinbodies. These data support a model which proposes that BiP associateswith the newly synthesized prolamine polypeptide to facilitateits folding and assembly into a protein inclusion body, andis then recycled. (Received October 21, 1996; Accepted January 20, 1997)     

Carbohydrate Metabolism in the Developing Endosperm of Rice Grains

The metabolism of carbohydrates in developing rice endospermwas characterized by a comparison of levels of activities of33 major enzymes between the endosperm and green leaves of rice.Activities of ADPglucose pyrophosphorylase, starch synthaseand branching enzyme (Q-en-zyme), compared on the basis of solubleprotein content, were markedly higher in endosperm than in greenleaves. The high levels of Q-enzyme may be responsible for theefficient production of starch in the rice endosperm. The measurementof levels of metabolic intermediates and the localization ofkey enzymes in isolated amyloplasts from rice endosperm supportthe view that sucrose is metabolized in the cytoplasm via thepathway: sucroseUDPglucosehexose-PFBPtriose-P. Triose-P thenenters the amyloplast, where it is converted to G1P via FBPand, finally, G1P is converted to starch by the concerted reactionsof ADPglucose pyrophosphorylase, starch synthase and Q-enzyme. ¹Present address: Yamagata Prefectural Agricultural Experi mentStation, Minorigaoka, Yamagata, 990-02 Japan. ²Present address: Institute of Biological Sciences, The University of Tsukuba, Tsukuba Science City, Ibaraki, 305 Japan. (Received February 15, 1989; Accepted June 10, 1989)     

水稻胚与胚乳分化发育中的内源多胺

稻胚发育过程中,其内源多胺以腐胺、亚精胺为主。在幼胚分化期,腐胺和亚精胺的含量很高;幼胚分化完成时,其含量急剧下降;直至分化后期才趋稳定。在胚及胚乳发育时期,还出现一种未知多胺X_(22),其含量除在胚分化完成时较少外,在胚发育的其他各期中,含量则一直很高。DNA和蛋白质含量的变化,从分化期开始递增直至物质积累成熟期,其趋势均相同。多胺可能参与胚与胚乳中核酸和蛋白质合成的调节。     

Isolation and Characterization of a Proteinaceous Protease Inhibitor from Bacillus subtilis

A proteinaceous protease inhibitor which might have an intracellular role in modulating protease activity during sporulation was isolated from B. subtilis IFO 3027 by trichloroacetic acid and ethanol precipitations, and column chromatographies on SP-Sephadex, DEAE-Sephadex, DE AE-Toyopearl and Sephadex G-75.

The molecular weight of the inhibitor was estimated by gel filtration and SDS polyacrylamide gel electrophoresis to be about 16,000. The isoelectric point was determined as pH 4.8. The inhibitor is an acid and thermostable protein. The-amino acid sequence in the amino terminal region was determined to be (Met)-Glu-Asn-Gln-Glu-Val-Val-Leu-X-X-Asp-Ala-Ile-Gln-Glu- ··· (X, unidentified).

In addition to cytoplasmic serine proteases of the inhibitor-producing strain, the inhibitor inhibits various microbial serine proteases.     

Studies on the Non-starchy Polysaccharides of the Endosperm of Buckwheat

Water soluble polysaccharides from the buckwheat endosperm was fractionated by salting out and a DEAE-cellulose column (phosphate form) chromatography and the main component (polysaccharide A₁) was isolated as an ultracentrifugally and electrophoretically pure preparation.

The content of polysaccharide A₁ in the buckwheat endosperm was 0.1~0.2%.

Its water solution showed high viscosity and [α]_d was +39.4°. The molecular weight was 240,000~260,000.

Polysaccharide A₁ consisted of xylose, mannose, galactose and glucuronic acid. The hydrolysis of methylated polysaccharide A₁ gave 2,3,4-tri-O-methyl-xylose, 2,3,4,6-tetra-O-methyl-galactose, 2,4,6-tri-O-methyl-galactose, di-O-methyl-mannose and 4-O-methyl- and 5-O-methyl-glucuronic acid. These results suggested that the main chain of this polysaccharide consisted of glucuronic acid, mannose and galactose and the former two occupied branching position with xylose and galactose residues as nonreducing end.     

两个水稻胚乳启动子的克隆及表达分析

启动子的克隆对基因表达及基因工程研究有重要意义。根据数据库中EST丰度,从水稻中克隆了两个预测在水稻胚乳中高效表达的启动子Os772和Os359,并将启动子片段与GUS报告基因融合,构建了重组表达载体。通过农杆菌介导方法将其导入水稻愈伤组织细胞。转基因水稻经GUS组织化学分析显示,Os772和Os359能启动GUS基因在水稻胚乳中表达但不能在根、茎、叶和花中表达。该结果表明Os772和Os359为两个水稻胚乳特异性启动子。     

Studies on Initiation of Apple Endosperm Callus and Variations of Chromosomal Ploidy on Callus Cells of Endosperm

The present paper deals with the initiation of endosperm callus of apple and the ploidy on callus cells through subcultures. According to our observations, the low capacity of differentiation has been discussed. The following conclusions are drawn: 1. The callus initiaed at 2–5 layer cells beneath epidermis. Some cells in this region changed into initials of endosperm callus and from initial masses then develop into cellular masses of endosperm callus protruding on the endosperm surface. The differentiation and growth of callus masses through subcultures may carry out in embryonal and transitional cellular masses in the peripheral zone and basal zone. 2. The ploidy in callus cells is very unstable. Through repeated subcultures the ploidy of the callus cells vary greatly. Most cells (about 80%) contain polyploid and a large number of aneuploid neuclei, while the triploid cells only constitute the minority (about 2.5%–3%). Hence the ploidy in callus cells produced by well developed endosperm vary greatly. 3. It is highly probable that the above mentioned variation of ploidy in callus cells of endosperm, particularly within the embryonal and transitional cellular masses is the cause of the low capacity of differentiation.     

Studies on the Non-starchy Polysaccharides of the Endosperm of Naked Barley

The F-1 β-glucan from naked barley endosperm, a main component of water soluble β-glucan, has been subjected to degradation with the laminarinase from Bacillus circulans. This enzyme converts the F-1 β-glucan to a trisaccharide, 3-O-β-cellobiosyl-d-glucose*, and a tetrasaccharide, 3-O-β-cellotriosyl-d-glucose*, as the main products. These products, which constitute 74% of the polymer, have been identified by chemical methods. As the minor or trace components, laminaribiose, cellobiose, cellotriose** and two unidentified tetrasaccharides are detected. Overall data show that F-1 β-glucan mainly consists of two types of structural sequences; one is trimeric unit in which a single β-(1→3) linkage alternates with two consecutive β-(1→4) linkage, and the other, a tetrameric unit in which a single β-(1→3) linkage alternates with three consecutive β-(1→4) linkages.

It is shown that this laminarinase from B. circulans hydrolyses the glucoside bond of the reducing side of 1,3-linked β-d-glucopyranose residues.     

Studies on the Non-Starchy Polysaccharides of the Endosperm of Naked Barley

Two β-glucans were prepared from the aqueous extract of the endosperm of naked barley. The extraction was carried out under conditions which obviated enzyme action and eliminated the contamination of starch fraction. The precipitate obtained with 20% concentration of ammonium sulfate of the extract was adsorbed on DEAE-cellulose (borate form) column and fractionated into two fractions by the elutions with H₂O and 0.5 N NaOH, respectively. From 5 kg. of the endosperm the yield of the former was 13.3 g. (0.27% of the endosperm), the latter was 2.0 g. (0.04%). Both showed only one peak ultracentrifugally, were slightly levorotatory, not degraded with α-amylase, and consisted of only d-glucose. These data suggest that they are both β-glucans.

β-Glucan consumed in periodate oxidation 0.74 mole periodate per 1 mole of anhydro- glucose. Hydrolysate of the methylated β-glucan gave 2, 3,6-tri-O-methyl-d-glucose and 2, 4, 6-tri-O-methyl-d-glucose, and methanolysate of the methylated β-glucan gave methyl 2, 3, 6-tri-O-methyl-β-d-glucopyranoside and methyl 2, 4, 6-tri-O-methyl-β-d-glucopyranoside, although α-anomers were not separated. From sedimentation and diffusion coefficients the molecular weight of the β-glucan was calculated as 220,000.

These results indicated that the β-glucan was consisted of β-1, 4 and β-1, 3 linked glucopyranose residues with proportions of 2.5 : 1 to 3:1 (β-1, 4 linkage to be predominant) and that the β-glucan had a linear structure.     

Studies on the Non-Starchy Polysaccharides of the Endosperm of Naked Barley

The structure of F-4 β-glucan, a minor component of water soluble non-starchy polysaccharides from the endosperm of naked barley, was elucidated. Hydrolysate of the methylated F-4 β-glucan gave 2,3,6-tri-O-methyl-d-glucose and 2,4,6-tri-O-methyl-d-glucose as main components with small amounts of 2,3,4,6-tetra-O-methyl-d-glucose and unidentified di-O-methyl-d-glucose. This result indicated that the main chain of F-4 β-glucan consisted of 1,4- and 1,3-linked β-d-glucopyranose residues with proportions of approximately 2:1 (β-1,4 linkage to be predominant) with some branching.     

Studies on the Non-Starchy Polysaccharides of the Endosperm of Naked Barley

F-1 β-glucan, the main component of water soluble non-starchy polysaccharides from naked barley endosperm, has been subjected to degradation with cellulases from Trichoderma viride and Trametes sanguinea. The former cellulase converted F-1 β-glucan to d-glucose, celiobiose, 4-O-β-laminaribiosyl-d-glucose and 4²-O-β-laminaribiosyl-cellobiose as main products. From the latter preparation four fractions of cellulase were separated. Their hydrolysing mechanisms against F-1 β-glucan differed from each other. Thus, it was suggested that the hydrolysing mechanism of cellulase was different when its origin or fraction in the same origin differed.

Cellulase from Trichoderma viride was almost similar to those from Streptomyces spearated by Parrish et al. and from Aspergillus niger by Stone et al. about the hydrolysing mechanism on barley β-glucans, though a small difference was found.     

Studies on the Development of the Embryo and Endosperm of Nitraria sibirica Pall

The pollen tube enters the embryo sac through the crassinucellus at the micropylar end, and brings about the porogamy. The embryogeny corresponds to the Solanad type. The defference of the suspensor structure is notable by comparing it with the other genera of Zygophyllaceae that have been studied. The endosperm is of the Nuclear type. Mitosis is the main form of the free endosperm nuclei proliferation. No cell plates develop in the early free nuclear division, however, they appear in late development, without developing into the cell wall and disappear ultimately. At the late globular embryo stage, cell formation in endosperm starts first from the micropylar end. The first anticlinal walls develop from the cell plate that is initiated from tile phragmoplast as a result of normal cytokinesis. Follwing this a wall begins to grow from the base of the cell plates, the outer growing margin soon fuses with the wall of the central cell, and the inner growing margin continues to grow towards the central vacuole. The growing walls branch and eventually fuse on the side nearest the central vacuole. Thus, the first periclinal walls are initiated, and a complete endosperm cell is formed. Along with the development of embryo, cell is gradually formed in the endosperm from the micropylar end towards the chalazal end, but the chalazal endosperm is still coenocytic until the endosperm disintegrate completely. The mature seed has no endosperm.     

粳稻胚乳细胞增殖与蔗糖代谢的关系

蔗糖代谢为水稻胚乳发育提供物质和能量。为明确二者的量化关系,本研究通过调节源库关系获得不同源供应水平下的代表性粒位籽粒,进而分析了蔗糖、果糖、葡萄糖及可溶性总糖的含量与胚乳细胞增殖的关系。结果表明,改变品种的源库比例（源大库小）,下部粒位籽粒胚乳细胞数目明显增加,总体上,可溶性总糖含量与细胞数目呈极显著负相关,与细胞增殖速率呈极显著正相关,高的蔗糖/葡萄糖、蔗糖/果糖有利于胚乳细胞数目增多。在细胞增殖前期（花后5d）,高葡萄糖、己糖含量有利于提高胚乳细胞数目,高葡萄糖含量还可提高细胞增殖速率。细胞增殖中后期（花后7d）,蔗糖、果糖和葡萄糖含量与胚乳细胞数目、增殖速率呈显著负相关。     

Studies on Endosperm Development and Deposition of Storage Reserves in Coix lacryma-jobi

The transition from free nuclear to cellular endosperm of Coix lacryma-jobi was eompleted 2 days after pollination. By 3 days after pollination the central cell was filled with endosperm cells. At first all cells of endosperm underwent division, later cell division was limited mainly in the peripheral region. 10 days after pollination the epidermal layer ceased its periclinal division and became the aleurone layer. Cell division persisted in the subepidermal 'cambium-like layers until the caryopsis nearly matured. Ceils of the inner region of endosperm became enlarged. Several layers of transfer cells were formed at the basal part of the endosperm. Starch grains appeared in endosperm cells on the 9th day after pollination. 10 days after pollination, lipid bodies occurred in the aleurone layer and the underlying layers. 13 and 15 days after pollination, the small vacuoles of aleurone cells contained protein and 20 days after pollenation they became aleurone grains. By 15 days after pollination pro tein bodies were formed in starch endosperm. Storage reserve deposition continued until the grain ripened. A correlation between endosperm and emoryo development was also observed.     

Studies on the Membrane of the Almond Endosperm Haustorium: With six Plates and one Figure in the Text

The endosperm of Primus amygdalus develops after fertilizationinto a long narrow tube reaching the chalaza. This tubular formationdoes not become cellular and functions as a haustorium. Cellularizationoccurs at the micropilar end of the endosperm and, after theseed has reached its definite size, the cellular endosperm growsat the expense of the nucellus pushing toward the chalaza thehaustorial portion. The membrane of the haustorium is mainly cellulosic and clearlylamellated as shown by electron micrographs of its sections.The X-ray diagrams show lack of orientation through the thicknessof the membrane and indicate some slight difference in spacingsbetween the haustorial cellulose and the cellulose of Cladophorarupestris An interesting feature of the haustorial membrane is the presenceof numerous roundish structures, about 4µ in diameter,which show an irregular maltese cross when observed under crossednicols. The electron micrographs show that these structurescorrespond to regions of the membrane where the regular lamellationis disturbed, the membrane appearing swollen and the interiorof the ‘bubble’ being occupied by irregular spiral-likecoils. In some cases, through stripping of the membrane, somerather thick coiled strands have been isolated.     

Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G_M1-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine.