Enzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinus communis L

The metabolism of sucrose to long chain fatty acids in the endosperm of developing castor bean (Ricinus communis L.) seeds requires a combination of cytosolic and proplastid enzymes. The total activity and the subcellular distribution of the intermediate enzymic steps responsible for the conversion of sucrose to pyruvate have been determined. Hexose phosphate synthesis from sucrose occurs in the cytosol along with the first oxidative step in the pentose phosphate pathway, glucose-6-phosphate dehydrogenase. The proplastids contain the necessary complement of glycolytic enzymes to account for the in vivo rates of acetate synthesis from glucose 6-phosphate. These organelles also contain the majority of the cellular 6-phosphogluconate dehydrogenase, transketolase, and transaldolase activities.     

Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)     

6-Phosphogluconate Dehydrogenase Isoenzymes from the Developing Endosperm of Ricinus communis L

The cytosolic and proplastid isoenzymes of 6-phosphogluconate dehydrogenase were purified from the developing endosperm of the castor bean (Ricinis communis L.). No differences in physical or kinetic properties were found for the purified isoenzymes. Each was composed of two identical 55,000 subunits. They had identical pH optima of 7.8 to 8.0 and similar MgCl₂ stimulation for the oxidative decarboxylation of 6-phosphogluconate. The Km values for 6-phosphogluconate were 12 and 9.6 micromolar and for NADP⁺ were 4.1 and 5.4 micromolar for the cytosolic and proplastid isoenzymes, respectively. Therefore, the synthesis of two distinct 6-phosphogluconate dehydrogenase isoenzymes does not appear to have any kinetic significance for the developing seed. However, changes in the proplastid contribution toward carbohydrate metabolism occur in the developing seed and may necessitate independent gene expression to allow for a unique and flexible subcellular distribution of isoenzymes during development.     

Temperature-induced Phase Changes in the Membranes of Glyoxysomes, Mitochondria, and Proplastids from Germinating Castor Bean Endosperm

Sucrose-gradient purified mitochondria, glyoxysomes, and proplastids from germinating castor bean (Ricinus communis L.) endosperm were examined by electron-spin resonance spectroscopy. A temperature-induced phase change was demonstrated in all of these organelles, their derivative membranes, and in micelles formed from the membrane phospholipids. The apparent transition temperature of the membrane lipids varied slightly between the samples, but in all cases, fell within the temperature range around 10 C where physiological and biochemical changes in the response to temperature for most chilling-sensitive plants occur.     

Isoenzymes of the Glycolytic and Pentose Phosphate Pathways in Proplastids from the Developing Endosperm of Ricinis communis L

Two isoenzymes each of hexose-P isomerase, aldolase and 6-P-gluconate dehydrogenase have been found in the endosperm of developing castor beans (Ricinus communis L.). One isoenzyme for each activity is present in the proplastid fraction. Only one form of glucose-6-P dehydrogenase was found. It is suggested that the partition of an enzyme activity between cytosol and plastid is regulated by the synthesis of isoenzymes which are subcellular site specific. In addition, this report describes the use of diethylaminoethyl-Sephadex A-25 sievorptive chromatography for the preparation of plant enzymes.     

蓖麻单性雌株组织培养和快速繁殖

1　植物名称　蓖麻 (Ｒｉｃｉｎｕｓｃｏｍｍｕｎｉｓ)。2　材料类别　单性雌株的顶芽、腋芽、带节的茎段。3　培养条件　 ( 1 )起始培养基 :1 /3ＭＳ 6 ＢＡ 0 .5ｍｇ·Ｌ- 1 (单位下同 ) ＩＢＡ 0 .0 1 ;( 2 )芽增殖培养基 :ＭＳ(改良 ) 6 ＢＡ 0 .5～ 0 .8 ＩＢＡ 0 .0 1～ 0 .0 3;( 3)生根培养基 :1 /2ＭＳ ＮＡＡ 0 .0 5 活性炭(适量 )。培养基中添加 3%蔗糖、0 .7%琼脂 ,ｐＨ5 .8。培养温度 2 4～ 2 7℃ ,光照 1 2～ 1 4ｈ·ｄ- 1 ,光照度约为 1 5 0 0ｌｘ。4　生长与分化情况4.1　无菌材料的获得　以蓖麻单性雌株的芽尖和带腋…     

Energy Requirement for the Import of Protein into Plastids from Developing Endosperm of Ricinus communis L

Leucoplasts isolated from developing endosperm of Ricinus communis L. will import the precursor of the small subunit of ribulose bisphosphate carboxylase from pea shoots and process it to its mature molecular weight (SA Boyle, SM Hemmingsen, DT Dennis [1986] Plant Physiol 81: 817-822). This process requires energy in the form of ATP. GTP, CTP, and UTP are inactive. ADP will also satisfy the energy requirement, probably through the action of adenylate kinase in the envelope. Fatty acid biosynthesis which occurs within these leucoplasts also requires ATP for maximal activity. Phosphoenolpyruvate will stimulate fatty acid biosynthesis approximately three times as effectively as ATP through the generation of ATP within the organelle by the action of the plastid pyruvate kinase. However, phosphoenolpyruvate under similar conditions will not stimulate the uptake of the small subunit of ribulose bisphosphate carboxylase into leucoplasts. These results indicate that ATP is required outside the leucoplast for protein uptake and that internally generated ATP is not effective in this process.     

Characterization of ATP-Dependent Fructose 6-Phosphate 1-Phosphotransferase Isozymes from Leaf and Endosperm Tissues of Ricinus communis

Plastid and cytosolic isozymes of ATP:fructose 6-phosphate 1-phosphotransferase (PFK_p and PFK_c, respectively) have been isolated from leaves and developing endosperm tissues of the castor oil plant (Ricinus communis L). Endosperm PFK_p has been purified to apparent homogeneity. Polyclonal antibodies raised against one of the four polypeptides associated with potato tuber PFK (molecular mass, 46 kilodaltons) immunoprecipitated developing endosperm and leaf PFK_p, but not PFK_c isozymes. Western blots, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and analytical gel filtration show that PFK_p from developing endosperm is a 220 kilodalton homotetramer composed of 57 kilodalton subunits. Kinetic studies of leaf PFK_p and PFK_c isozymes reveal both similarities and differences to the characteristics of the respective endosperm isozymes studied previously (WJ Garland, DT Dennis [1980] Arch Biochem Biophys 204: 302-317). The immunological and kinetic data suggest that leaf and developing endosperm PFK_p are different but structurally related proteins.     

Graviresponsiveness and the Development of Columella Tissue in Primary and Lateral Roots of Ricinus communis

Half-tipped primary and lateral roots of Ricinus communis cv Hale bend toward the side of the root on which the intact half-tip remains. Therefore, the minimal graviresponsiveness of lateral roots is not due to the inability of their caps to produce growth effectors (presumably inhibitors). The columella tissues of primary (i.e. graviresponsive) roots are (a) 4.30 times longer, (b) 2.95 times wider, (c) 37.4 times more voluminous, and (d) composed of 17.2 times more cells than those of lateral roots. The onset of positive gravitropism by lateral roots is positively correlated with a (a) 2.99-fold increase in length, (b) 2.63-fold increase in width, and (c) 20.7-fold increase in volume of their columella tissues. We propose that the minimal graviresponsiveness of lateral roots is due to the small size of their columella tissues, which results in their caps being unable to (a) establish a concentration gradient of the effector sufficient to induce gravicurvature and (b) produce as much of the effector as caps of graviresponsive roots.     

胚乳中淀粉的合成

禾本科植物胚乳内所含有的淀粉根据其结构、组成可以分为两类：直链淀粉（由α-1,4糖苷键连接的多聚D-葡萄糖）和支链淀粉（在以α-1,4糖苷键连接的主链上通过形成α-1,6糖苷键引入支链的多聚D-葡萄糖）。前者是以一种线性无序状态存在,而支链淀粉则是构成淀粉半晶体结构的主要成分。其中,除了负责合成作为糖基直接供体的ADP—Glc的酶AGPase外,直链淀粉中链的延伸反应由GBSSI完成,而支链淀粉的合成则相对复杂,需要SS、SBE、DBE、SP等一些酶的协同调控来共同完成。本文综述了胚乳中淀粉合成过程中所涉及的一些关键酶的研究进展,并对此研究领域进行了展望。     

Expression of the Genes for the alpha- and beta-Subunits of Pyrophosphate-Dependent Phosphofructokinase in Germinating and Developing Seeds from Ricinus communis

Various tissues from both germinating and developing castor seeds (Ricinus communis L.) have been analyzed for the level of expression of the genes for the α- and β-subunits of pyrophosphate-dependent phosphofructokinase (PFP). In tissues in which PFP is expressed, there is a single mRNA species of approximately 2 kilobases for each of the subunits. In germinating endosperm, the gene for the α-subunit is expressed at an earlier time after imbibition than that for the β-subunit, whereas in developing castor seed endosperm, both genes are highly and coordinately expressed. During seedling development, there is tissue-specific expression of the two genes. Tissues in which there is a high level of mRNA correspond with tissues in which both subunits of PFP can be detected. The differential expression of the two subunit genes in germinating endosperm does not result in the presence of the α-subunit polypeptide in the absence of the β-subunit polypeptide. Southern analysis of castor genomic DNA indicates the presence of a single gene for both the α- and β-subunits of PFP in contrast with potato, in which there are at least two genes for each subunit.     

Studies of Glycollate Utilization and Some Associated Enzymes of C1 Metabolism in the Endosperm of Ricinus communis L.

Glycollate metabolism in 5-day-old endosperm tissues of Ricinuscommunis L. was examined by feeding micromolar quantities of[2-¹⁴C]glycollate to tissue slices. It was found that glycollatecarbon was rapidly incorporated into glyoxylate, glycine, serine,and carbon dioxide. Only small amounts of ¹⁴C were incorporatedinto the sugars. Changes in the distribution of ¹⁴C with timesuggested that glyoxylate was a primary product and that glycineand serine were secondary products of glycollate metabolism.The results of feeding experiments are interpreted as indicatingthat a glycollate pathway leading to sugar biosynthesis is ofminor importance compared to the rapid utilization of glycollatefor the biosynthesis of glycine and serine. Enzymes necessaryto catalyse the incorporation of glycollate into glycine andserine have been examined in castor-bean endosperm extracts.These included: glycollic acid oxidase, gloxylic acid reductase,glyoxylate transaminase, N¹⁰ formyltetrahydrofolate synthetase,N⁵,N¹⁰-methylenetetrahydrofolate dehydrogenase, and serine hydroxymethyltransferase.     

Transverse Distribution of Phospholipids in Organelle Membranes from Ricinus communis L. var. Hale Endosperm: MITOCHONDRIA AND GLYOXYSOMES

Phospholipase A₂ (Naja naja), the nonpenetrating dye trinitrobenzene sulfonate, and the penetrating dye dinitrofluorobenzene, were used to determine the transmembrane distributions of phospholipids of mitochondria and glyoxysomes isolated from endosperm tissue of castor bean (Ricinus communis L. var. Hale). These studies indicated that the phospholipid distributions were distinctly asymmetric in the accessible (reacted with the probes without total membrane disruption by detergents) pools of the glyoxysomal and inner mitochondrial membranes, but more nearly symmetric in the outer mitochondrial membrane. However, significant quantities of the phospholipids of the mitochondrial membranes were inaccessible to the probes used. An increased accessibility of the phospholipids of all membranes following Triton X-100 dispersion was found, and protein to phospholipid ratios in organelle membranes were found to correlate inversely with the accessibility of the phospholipids to the probes. The inaccessible phospholipids may be involved in lipid-protein interactions.     

Cytokinins in the vascular saps of Ricinus communis

Roots are recognised as the major sites of cytokinin synthesis and shoots receive a continuous supply of cytokinins from the roots. Although reports are available on the xylem mobility of putative free bases and their ribosides, relatively few studies on the phloem mobility of cytokinins have been reported. The origin of phloem-mobile cytokinins is uncertain but there is evidence which implicates a recirculation from the root source. This study is the first report in which zeatin and zeatin riboside from the root pressure exudate and phloem sap of Ricinus have been identified by full-scan GC-MS and quantified by GC-MS selective-ion-monitoring. In this study, the concentration of cytokinins in root pressure exudate was similar, but lower, and in the phloem sap higher than that reported previously. The concentration of cytokinins quantified in the phloem sap confirms their transport in the sieve tubes. The relatively high concentration of zeatin riboside detected in the root pressure exudate and of zeatin detected in the phloem sap indicate a possible vascular recirculation of these hormones.     

Purification and characterization of Ricinus communis invertase

An invertase from Ricinus communis leaves was purified 4,400-fold. The preparation was homogeneous by criteria of gel electrophoresis, gel permeation, adsorption, and ionic exchange chromatography. One optimum pH at 3.5 was observed with crude invertase; however, purified preparations showed two optima, at pH 3.5 and 5.5. Addition of bovine serum albumin restored one maximum at pH 3.5 and elicited a 30% activation of the invertase. The effect was caused by many other proteins and by heparin, dextran sulfate, and polyvinylpyrrolidone. Fructose, fructose 1,6-diphosphate, maleic, trans-aconitic, malic, and ascorbic acids were simple competitive inhibitors of the purified enzyme. Glucose was a noncompetitive inhibitor. The activation by proteins suppressed these inhibitory effects. The minimum concentration of activator necessary to reach the maximal activation or "point of optimal activation" was always reached at a concentration of 1 X 10(-6) M, independently of the nature of the activator, when 8.6 X 10(-12) mol of enzyme were used. Apparent molecular weight determinations of the enzyme in the presence and absence of activator and molecular weight determinations based on determinations of the point of optimal activation suggested that the purified enzyme is a heptamer (Mr of 77,900, Stokes radius 32 A, frictional ration f/fo 1.1, partial specific volume 0.749 ml/g) and that the activated form is a trimer consisting of two enzyme subunits and one activator molecule. The activation was lost by dilution of the trimer. The enzyme subunit, as isolated by gel filtration in the presence of sodium dodecyl sulfate (Mr 11,000) was inactive but quickly regained activity upon removal of sodium dodecyl sulfate.     

Cytokinin Biosynthesis in Germinating Maize Embryos

Three-day-old germinating embryos of Zea mays were incubatedwith [8-¹⁴C]adenine for 8 and 24 h. Use of high performanceliquid and thin layer chromatography provided evidence for theincorporation of adenine by isolated germinating embryos intotrans-zeatin. These results are at variance with similar labellingstudies and possible reasons for this discrepancy are discussed. Key words: Adenine, germination, maize kernels, zeatin     

Potassium Uptake and Transport in Roots of Ricinus communis

The uptake, accumulation, and transport of potassium by excisedroots of Ricinus communis were investigated. It was found that: 1. The transport of potassium to the vessels was closely dependenton the supply of the ion in the medium. 2. The flux of potassium to the vessels was only a fractionof the total potassium taken up and 99 per cent, of that absorbedwas accumulated in the root tissues. Once retained in this waypotassium appeared to become unavailable for transport to thevessels. 3. There was a fixed relationship between the potassium concentrationsof the medium and the exudate when potassium was supplied aspotassium nitrate in the medium at a range of concentrations.This relationship was altered by the presence of other ionsin the medium.     

Protein Body Degradation in the Starchy Endosperm of Germinating Sorghum

Taylor, J. R. N., Novellie, L. and Liebenberg, N. v. d. W. 1985.Protein body degradation in the starchy endosperm of germinatingsorghum.—J. exp. Bot. 36: 1287–1295. Transmission electron micrographs of starchy endosperms of germinatingsorghum indicate that the protein bodies are degraded predominantlyby progressive hydrolysis of prolamin from their surface. Theappearance of holes within partially degraded protein bodiesindicates that some internal hydrolysis also takes place. Chemicalanalyses of protein bodies isolated at different stages duringgermination showed that their amino acid composition and electrophoreticpattern remained relatively unchanged during hydrolysis. Theend result of protein body degradation was that the organellescompletely disappeared leaving empty starchy endosperm cells.The protein bodies did not swell prior to or during degradation.This mode of protein body degradation differs from that in germinatingdicotyledonous seeds and in the aleurone layer and embryo ofcereal seeds but was identical to the mode of prolamin proteinbody degradation in the starchy endosperm of germinating riceseeds. Key words: Sorghum bicolor, protein body degradation, prolamin