Expressed Sequence Tags from Developing Castor Seeds

To expand the availability of genes encoding enzymes and structural proteins associated with storage lipid synthesis and deposition, partial nucleotide sequences, or expressed sequence tags (ESTs), were obtained for 743 cDNA clones derived from developing seeds of castor (Ricinus communis L.). Enrichment for seed-specific cDNA clones was obtained by selecting clones that did not detectably hybridize to first-strand cDNA from leaf mRNA. Similarly, clones that hybridized to storage proteins or other highly abundant mRNA species from developing seeds were selected against. To enrich for endomembrane-associated proteins, some clones were selected for sequencing by immunological screening with antibodies prepared against partially purified endoplasmic reticulum membranes. Comparison of the deduced amino acid sequences of the ESTs with the public data bases resulted in the assignment of putative identities of 49% of the clones selected by differential hybridization and 71% of the clones selected by immunological screening. Open reading frames in 100 of the ESTs exhibited higher homology to 78 different nonplant gene products than to any previously known plant gene product.     

Glucose-6-Phosphate Dehydrogenase in Plastids from Developing Castor Bean Seeds

Glucose-6-phosphate dehydrogenase, together with the other enzymesof pentose phosphate pathway, was found in the cytosol as wellas in the plastid from developing castor bean (Ricinus communisL.) seeds. The plastid enzyme was found in both the matrix andthe membrane. The plastid enzyme has a sharp pH profile withthe optimum at 8.5, while the cytosolic enzyme has a broad pHprofile, optimum at 7.5. The plastid enzyme was inactivatedby storage at 0°C and by detergents such as Triton X-100,Brij and Nonidet, but the cytosolic enzyme was not. Slab geldisc electrophoresis indicated that three isoenzymes of glucose-6-phosphatedehydrogenase were found in the plastid but one enzyme in thecytosol of developing castor bean seed. From the presence ofglucose-6-phosphate dehydrogenase in the plastid, the operationof whole pentose phosphate pathway in this organelle of developingcastor bean seeds is suggested. (Received September 21, 1982; Accepted January 17, 1983)     

The Effects of Gibberellic Acid on Organelle Biogenesis in the Endosperm of Germinating Castor Bean Seeds

Mature seeds of castor bean (Ricinus communis L.) were imbibedin tap water or 0.3 mM GA₃, planted in vermiculite moistenedwith tap water or 0.3 mM GA₃, and incubated at 32 ?C. Duringthe course of germination, GA₃ promoted marked increases inthe activities of the glyoxysomal marker enzyme isocitrate lyaseand certain mitochondrial marker enzymes, but did not affectthe ER marker enzyme choline phosphotransferase. Glyoxysomaland ER protein and phospholipid were not increased in amountby GA₃, whereas mitochondrial protein and phospholipid were.SDS-polyacrylamide gels of the glyoxysomal matrix polypeptidesfrom GA₃-treated beans exhibited two polypeptides additionalto those found to be common to both GA₃-treated and controltissue. Incorporation of CDP-(methyl ¹⁴C)-choline into intactendosperm tissue and the distribution amongst the glyoxysomes,mitochondria, and ER of newly synthesized phosphatidyl-(methyl¹⁴C)-choline was unchanged by GA₃.     

Phospholipids and the Phospholipid Fatty Acids of Germinating Hazel Seeds (Corylus avellana L.)

An analysis of the phospholipids and phospholipid fatty acidsof germinating hazel seeds has been carried out. The phospholipidcontent of the cotyledons and embryonic axes increased duringgermination. Early increases in the relative amounts of phosphatidylglycerol and decreases in the relative amounts of phosphatidylethanolamine occurred in both cotyledons and axes. The laterstages of germination were accompanied by further changes inthe phospholipid composition of the axes. Increases in the degreeof unsaturation of the C₁₈ fatty acids occurred in both tissues.     

Accelerated Germination of Maize Seeds Under Chilling Stress by Osmotic Priming and Associated Changes in Embryo Phospholipids

Osmotic priming of maize seeds (Zea mays L. cv. Partap) usingpolyethylene glycol or potassium salts (K₂HPO₄, KH₂PO₄, KNO₃and K₂HPO₄+KNO₃) resulted in accelerated germination at a chillingtemperature (10 °C). The response of seeds primed in solutionsof either 2.5% K₂HPO₄ or 2.5% K₂HPO₄+ KNO₃ was particularlymarked compared with the untreated seeds, and the effect ofpriming was largely retained after seeds had been dried back.All embryo phospholipid fractions and sterols increased duringsalt-priming and the proportion of phospholipid which was diphosphotidylglycerol(DPG) also increased. It is suggested that the marked increasein the DPG content of primed embryos may be due to enhancedinternal organization of their mitochondrial membranes, andthat the benefit of osmotic priming may be at least partly dueto an increased potential for ATP accumulation. Germination, Zea mays L., osmotic priming, phospholipid changes     

Phospholipids in myofibrils

The myofibrils were shown to be lipoprotein structures. Lipoprotein nature of these structures was confirmed by their lipid phosphorus to protein ratio in a completely dispersed myofibril preparation of the skeletal muscle cells. The ratio was found to be 49.65 ± 3.95 microgram P / mg protein. The phospholipids present in the myofibril preparation were determined to be phosphatidylcholine and phosphatidylethanolamine.     

Suborganellar Localization and Molecular Characterization of Nonproteolytic Degraded Leukoplast Pyruvate Kinase from Developing Castor Oil Seeds

Several genes involved in the ability of Synechococcus sp. PCC 7942 to grow under different CO2 concentrations were mapped in the genomic region of rbcLS (the operon encoding the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase). Insertion of a cartridge encoding kanamycin resistance within open reading frame (ORF) 78, designated ccmJ, located 7 kb upstream of rbcLS, resulted in a kanamycin-resistant, high-CO2-requiring mutant, M3, which does not contain normal carboxysomes. ccmJ shows significant homology to csoS1 encoding a carboxysomal shell polypeptide in Thiobacillus neopolitanus. Analysis of the polypeptide pattern of a carboxysome-enriched fraction indicated several differences between the wild type and the mutant. The amount of the ribulose-1,5-bisphosphate carboxylase/oxygenase subunits was considerably smaller in the carboxysomal fraction of the mutant when compared to the wild type. On the basis of the sequence analyses, ORF286 and ORF466, located downstream of ccmJ, were identified as chlL and chlN, respectively, which are involved in chlorophyll biosynthesis in the dark.     

蓖麻生物工程研究进展

蓖麻是一种高蓄能植物和工业原料植物,具有很大的开发利用价值。本文从组织培养和遗传转化两个方面并结合本实验室的工作综述了蓖麻生物工程研究的最新进展。在蓖麻组织培养方面,不同的外植体中以成熟种子的胚轴最适宜,而在不同激素中以TDZ诱导丛生芽的效率最高,并以此为基础建立了蓖麻离体再生体系。在遗传转化方面,不同的转化方法中以农杆菌介导法最适合蓖麻转化。蓖麻胚轴对卡那霉素不敏感,潮霉素是蓖麻转化的适宜筛选剂。文中指出了蓖麻生物工程研究中存在的问题,并对应用生物技术培育蓖麻新品种和促进蓖麻生产的可能性进行了讨论。     

Phospholipids in Mediterranean cephalopods

Polar lipids of the cephalopods Eledone moschata, Sepia officinalis and Todarodes sagittatus mantle, represent 50.5%, 66.1% and 74.2% of wet tissue respectively. On the other hand the polar lipids of these three species of cephalopods constitute of 80.8%, 94.8% and 93.7% of phospholipids, respectively. The main phospholipids identified were phosphatidylcholine (52.2, 51.3 and 58.4% of total phospholipids respectively in the above mentioned species), phosphatidylethanolamine (18.1, 19.7 and 23.9%), sphingomyelin (10.7, 15.2 and 6.7%), lyso-phosphatidylcholine (3.1, 3.8 and 1.8%) and the unusual lipid ceramide aminoethylphosphonic acid (15.9, 10 and 9.2%). The 56.8% of phosphatidylcholine in Eledone moschata, the 46% in Sepia officinalis and the 74.1% in Todarodes sagittatus refer to the structure of 1,2-diacyl-glycerocholine and the remaining percentage refer to the structure of 1-o-alkyl-2-acyl-glycerocholine or 1-o-alkyl-1-enyl-2-acyl-glycerocholine. The 87.2% of phosphatidylethanolamine in Eledone moschata, the 81% in Sepia officinalis and the 90.7% in Todarodes sagittatus refer to the structure of 1,2-diacyl-glyceroethanolamine and the remaining percentage refer to the structure of 1-o-alkyl-2-acyl-glyceroethanolamine or 1-o-alkyl-1-enyl-2-acyl-glyceroethanolamine. The major saturated fatty acids in phosphatidylcholine and phosphatidylethanolamine were C16:0 (30.3-67.5% and 23.2-54.5%) and C18:0 (3.6-17% and 15.4-28%), respectively, while the major unsaturated fatty acids in these lipids were C18:1n-9, n-7 (1.0-7.3% and 5.3-10.5%), C20:5n-3 (1.5-9.8% and 4,5-15.8%) and C22:6n-3 (12.5-42.0% and 7.0-11.3%), respectively.     

Leucoplast Pyruvate Kinase from Developing Castor Oil Seeds : Characterization of the Enzyme's Degradation by a Cysteine Endopeptidase

Leucoplast pyruvate kinase (PK_p; EC 2.7.1.40) from endosperm of developing castor oil seeds (Ricinus communis L. cv Baker 296) appears to be highly susceptible to limited degradation by a cysteine endopeptidase during the purification of the enzyme or incubation of clarified homogenates at 4°C. Purified castor seed PK_p was previously reported to consist of immunologically related 57.5 and 44 kilodalton subunits (Plaxton WC, Dennis DT, Knowles VL [1990] Plant Physiol 94: 1528-1534). By contrast, immunoreactive polypeptides of about 63.5 and 54 kilodaltons were observed when a western blot of an extract prepared under denaturing conditions was probed with affinity purified rabbit anti-(castor seed PK_p) immunoglobulin G. Proteolytic activity against PK_p was estimated by the disappearance of the 63.5 and 54 kilodalton subunits and the concomitant appearance of lower molecular mass immunoreactive degradation products during the incubation of clarified homogenates at 4°C. The presence of 2 millimolar dithiothreitol accelerated the degradation of PK_p. The conservation of the 63.5 and 54 kilodalton subunits was observed after extraction of the enzyme in the presence of 1 millimolar p-hydroxymecuribenzoate, or 1 millimolar Nα-p-tosyl-l-lysine chloromethyl ketone, or 10 millimolar iodoacetate. These results reveal that a cysteine endopeptidase was responsible for the in vitro proteolysis of PK_p. This endopeptidase is present throughout all stages of endosperm development. Its PK_p-degrading activity, however, appears to be most pronounced in preparations from older endosperm. When lysates of purified leucoplasts were incubated at 4°C for up to 21 hours, no degradation of PK_p was observed; this indicated an extra-leucoplastic localization for the cysteine endopeptidase. Although the in vivo subunit structure of PK_p remains uniform throughout all stages of endosperm development, the large decrease in PK activity that accompanies castor seed maturation coincides with a marked reduction in the concentration of PK_p.     

Fat Metabolism in Higher Plants. XXXVII. Characterization of the beta-Oxidation Systems From Maturing and Germinating Castor Bean Seeds

In the maturing castor bean seed (Ricinus communis), maximum β-oxidation appears at 28 days after flowering and in the germinating seed, 4 days after germination. Highest specific activities for both β-oxidation systems and their component enzymes are associated with cytosomal particles banding at a density of 1.25 g/ml in a sucrose gradient. Substrate specificity studies indicate that of several fatty acids, ricinoleate is oxidized most rapidly by the preparation from the maturing seed (28 days after flowering) while palmitate and linoleate are oxidized most rapidly by extracts obtained from tissue germinated for 4 days. The β-oxidation activities observed in both systems reflect the expression of activity of at least 3 of the component enzymes, crotonase, β-hydroxyacyl dehydrogenase and β-keto-thiolase, which rise and fall co-ordinately. Acyl thiokinase does not appear to play a limiting role in regulating β-oxidation per se under the conditions employed here.     

Direct Transesterification of Castor and Jatropha Seeds for FAME Production by Microwave and Ultrasound Radiation Using a SrO Catalyst

In the present study, we report on an optimized method for fatty acid methyl esters (FAME) production from castor and jatropha seeds. In order to identify the most effective biodiesel production method, we have compared three two-stage methods, each consisting of oil extraction (the first step) and FAME production by transesterification (the second step), with the same three techniques each conducted in one stage, i.e., direct transesterification. The three techniques are conventional heating, sonochemistry, and microwave radiation. The FAME product was analyzed by ¹H NMR spectroscopy and GC-MS. The SrO catalyst was reused successfully, together with seeds containing oil residues, for 10 cycles. The highest yield of FAME, 57.2?% of the total weight of the castor seeds, and a conversion of castor oil to FAME of 99.95?% were achieved in a one-stage method lasting 5?min using microwave radiation as a heat source. Using jatropha seeds leads to a yield of 41.1?% and a 99.7?% conversion of triglyceride to FAME under microwave irradiation in a one-stage method. The direct transesterification by sonication resulted in yields of 48.2?% and 32.9?%, and a 93.6?% conversion from castor and jatropha seeds, respectively.     

Differentiation of Castor fiber and Castor Canadensis by noninvasive molecular methods

The aim of the study was to develop a simple and reliable method for differentiation of the two phenotypic, very similar Eurasian and North American beavers. Hair bulbs were plucked as tissue samples from the fur of living animals. The mitochondrial cytochrome b locus was amplified by polymerase chain reaction and sequenced. The fragments of the two species differed at 44/41 nucleotide sites. RsaI recognised two mutations, resulting in a restriction fragment length polymorphism that seems to be species specific, as could be revealed by the banding pattern. Zoo Biol 19:511-515, 2000. Copyright 2000 Wiley-Liss, Inc.     

Phospholipids in the cytochrome oxidase reaction

Phospholipids and Emasol activate cytochrome oxidase by increasing its affinity for its substrate, cytochromec. Cardiolipin was most effective in activating cytochrome oxidase among phospholipids tested. Prior formation of a cytochromec-cytochrome oxidase complex changes the effect of phospholipids. In addition to their structural role in the last segment of the electron transport system, phospholipids can protect the enzyme from heat treatment and mercurial inhibition. They facilitate the interaction between cytochrome oxidase and cytochromec, as well as the cytochromec analogue, protamine.