Article

Isolation of Intact Plastids from Protoplasts from Castor Bean Endosperm

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Protoplasts were prepared from castor bean (Ricinus communis) endosperm by treatment with a mixture of the commercial enzymes Macerozyme R-10 and Cellulose "Onozuka" R-10. The protoplasts were gently ruptured by forcing the suspension through a hypodermic needle and the homogenate centrifuged on a linear sucrose gradient. From such a homogenate the mitochondria are recovered at their typical isopycnic density of 1.18 g/ml, but the glyoxysomes are retained, with other membranes, at a density of 1.13. The plastids reach their typical density of 1.22 on the gradient and are thus clearly separated from other organelles. Moreover, since essentially all of the ribulose bisphosphate carboxylase activity on the gradient is present in this fraction it can be concluded that the plastids are intact and have been recovered in high yield.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Protoplasts can be obtained from the leaves and callus of castor (Tudses et al. 2014). However, since the first protoplasts were isolated from castor bean endosperm in 1978 (Nishimura and Beevers 1978), only one group has applied castor protoplasts for use in gene expression, and the transfection frequency was 12.37% (Liu et al. 2019). ...
Article
Full-text available
Castor bean is an oil crop plant (Euphorbiaceae) found across the tropical, subtropical, and temperate regions. Despite its important oil properties and cultivation in a wide range of environments, the molecular mechanisms of castor’s adaptation and metabolism have not been fully clarified due to difficulties in genetic modification approaches. The protoplasts of several other plant species have been used as versatile cell-based model systems to elucidate the biological functions of genes and proteins. Here, we report an optimized protocol for protoplast isolation from the leaves and cotyledons of castor bean. The main parameters evaluated to achieve the maximum protoplast yield were the application of a cell wall-degrading enzyme solution, the osmotic pressure of the enzymolysis solution, and the enzymolysis time. Transient expression and the main influencing factors were validated by fluorescence microscopy of castor protoplasts. Our results suggest that castor protoplasts can be used as a productive cell-based system to explore the mechanisms involved in the molecular, biochemical, and functional characterization of castor bean genes.
... In this respect, the grape skin tissue closely resembles other, high sugar-containing, tissues such as beet roots, which also require high osmolarity medium for the production of stable protoplasts and vacuoles (14). The protoplasts produced from the grape skin did not release intact vacuoles upon treatment with 0.2 M Na4P207-HCl (pH 8.0) (26), upon dilution of the osmoticum (20), by polybasic DEAE-dextran treatment (10), or by shearing forces created upon uptake and expulsion in a syringe (18). ...
Article
Full-text available
Enzymic treatment of mature, anthocyanin-containing grape berry sub-epidermal tissues from DeChaunac grapes released intact protoplasts. Filtration of the protoplast suspension through glass wool under mild suction resulted in the release of vacuoles. The total and individual contents of anthocyanins, flavonol glycosides, hydroxycinnamic acid esters, sugars, organic acids, and cations were determined in both tissue and vacuole preparations. A method for pH determination in intact vacuoles is reported. Based on the qualitative and quantitative anthocyanin composition, the average pH of the vacuoles was determined to be 2.7 (sd +/- 0.17). The data suggest that anthocyanins are present in fruit subepidermal tissues in a noncomplexed form.
... This kind of preparation is necessary for the accurate investigation of nucleic acids, membrane lipids, protein and enzymes 11 . Earlier preparations of plastids include aqueous methods involving linear or discontinuous sucrose gradient centrifugation of mechanically prepared crude plastids 12 and from broken protoplasts 13,14 . But all these methods have problems regarding purity, and intactness and this have been accounted because of the lack of information on root plastid permeability 15 even though purification of intact chloroplasts and peroxisomes following centrifugation through a silica-sol pad or gradient has been reported in lots of previous studies 16 . ...
... Homogenized material or crude organelle preparations are applied to density gradients, and fractions are collected after centrifugation. The distribution profiles of organelles in these partially enriched fractions are characterized, for example, by assays of organelle-specific enzymes (6,7). Proteins displaying a distribution profile similar to those of organelle marker proteins can then be assigned to this subcellular compartment. ...
Article
Full-text available
Non-aqueous fractionation is a technique for enrichment of different subcellular compartments derived from lyophilised material. It was developed to study the subcellular distribution of metabolites. Here we analyse the distribution of about 1000 proteins and 70 metabolites, including 22 phosphorylated intermediates in wild-type Arabidopsis rosette leaves, using non-aqueous gradients divided into 12 fractions. Good separation of plastidial, cytosolic and vacuolar metabolites and proteins was achieved but cytosolic, mitochondrial and peroxisomal proteins clustered together. There was considerable heterogeneity in the fractional distribution of transcription factors, ribosomal proteins and subunits of the vacuolar-ATPase, indicating diverse compartmental location. Within the plastid, sub-organellar separation of thylakoids and stromal proteins was observed. Metabolites from the Calvin-Benson cycle, photorespiration, starch and sucrose synthesis, glycolysis and tricarboxylic acid cycle grouped with their associated proteins of the respective compartment. Non-aqueous fractionation thus proved a powerful method for the study of the organellar, and in some cases sub-organellar, distribution of proteins and their association with metabolites. It remains the technique of choice for assignment of subcellular location to metabolites in intact plant tissues and thus the technique of choice for doing combined metabolite-protein analysis in the same tissue sample.
... Our initial efforts to isolate rapeseed embryoplasts focused on these procedures although SDS-PAGE and MudPIT analysis of these plastid preparations indicated a high level of contamination with PSV and mitochondria proteins (data not shown). Protoplast isolation followed by cell lysis is reportedly milder than mechanical methods and was previously employed to isolate recalcitrant organelles including amyloplasts from maize endosperm [19], protein bodies from mung bean [20], and plastids from castor bean endosperm [21]. We present a modified procedure here for rapeseed embryos. ...
Article
Plastids are functionally and structurally diverse organelles responsible for numerous biosynthetic reactions within the plant cell. Plastids from embryos have a range of properties depending upon the plant source but compared to other plastid types are poorly understood and therefore, we term them embryoplasts. Isolating intact plastids from developing embryos is challenging due to large starch granules within the stroma and the prevalence of nonplastid, storage organelles (oil bodies and protein storage vacuoles) which compromise plastid integrity and purity, respectively. To characterize rapeseed embryoplasts it was necessary to develop an improved isolation procedure. A new method is presented for the isolation of intact plastids from developing embryos of Brassica napus seeds. Intactness and purity of embryoplast preparations was determined using phase-contrast and transmission electron microscopy, immunoblotting, and multidimensional protein identification technology (MudPIT) MS/MS. Eighty nonredundant proteins were identified by MudPIT analysis of embryoplast preparations. Approximately 53% of these proteins were components of photosystem, light harvesting, cytochrome b/f, and ATP synthase complexes, suggesting ATP and NADPH production are important functions for this plastid type.
Article
Vacuoles were prepared from endosperm tissue of 4-day-old castor bean seedlings (Ricinus communis var. Hale) and purified on a stepped sucrose gradient. It was shown by assays of marker enzymes that there was only trace contamination of the final preparation by other organelles (mitochondria, glyoxysomes, nuclei, spherosomes, and plastids) and by cytoplasmic components. Hydrolytic enzymes (acid protease, carboxypeptidase, phosphodiesterase, RNAase, phytase and beta-glucosidase) were present in the isolated vacuoles in amounts indicating a primarily vacuolar localization in vivo. The vacuoles also contained storage protein and high concentrations of sucrose. The over-all results indicate that the vacuoles from castor bean endosperm are the site of hydrolysis of the constituents of the protein bodies and are a temporary storage compartment for the sucrose produced from fat and protein reserves.
Article
Full-text available
Protoplasts were prepared from exponentially growing cell suspension cultures derived from Pinus taeda seedlings. The cell suspension was derived from callus that had been initiated on solid Murashige and Skoog medium supplemented with 2mg/1 2,4-D, and maintained in liquid LM medium (D.C. Verma et al, Proceedings of the 5th International Congress of Plant Tissue and Cell Culture, Tokyo, Japan, 1982, p. 59–60). Evaluation of a range of enzyme formulations showed that almost all cells could be converted to viable protoplasts in 9 hours with the combination of 0.5% each of Onozuka R-10, Macerozyme R-10, Hemicellulase, Driselase, and bovine serum albumin in osmoticum (0.4 M mannitol, 0.2 M sorbitol, 1.5 mM CaCl2, 0.7 mM KCl). Protoplast purification required only simple washing which was achieved without damaging centrifugation. When cultured in LM medium supplemented with 1.5mM CaCl2, 10 mM arginine, 10mM glutamine and 5 mM asparagine, cell wall formation was observed after 48 hours. The cultured protoplasts produced numerous colonies in 3 weeks which continued to grow in liquid medium.
Article
Full-text available
Intact amyloplasts from endosperm of developing wheat grains have been isolated by first preparing the protoplasts and then fractionating the lysate of the protoplasts on percoll and ficoll gradients, respectively. Amyloplasts isolated as above were functional and not contaminated by cytosol or by organelles likely to be involved in carbohydrate metabolism. The enzyme distribution studies indicated that ADP-glucose pyrophosphorylase and starch synthase were confined to amyloplasts, whereas invertase, sucrose synthase, UDP-glucose pyrophosphorylase, hexokinase, phosphofructokinase-2 and fructose-2,6-P2ase were absent fro the amyloplast and mainly confined to the cytosol. Triose-P isomerase, glyceraldehyde-3-P dehydrogenase, phosphohexose isomerase, phosphoglucomutase, phosphofructokinase, aldolase, PPi-fructose-6-P-1 phosphotransferase, and fructose-l,6-P2ase, though predominantly cytosolic, were also present in the amyloplast. Based on distribution of enzymes, a probable pathway for starch biosynthesis in amyloplasts of developing wheat grains has been proposed.
Article
The effects of ethanol concentration on growth, solute leakage, sugar absorption, and respiration in coleoptiles and roots of rice, oat, and wheat seedlings were measured and compared with the effects of removing O2. Whereas the responses to anoxia were immediate the responses to ethanol were time dependent and all required concentrations many fold those known to accumulate in N2. Thus the adverse effects of anoxia cannot be ascribed to ethanol accumulation per se. In most respects the rice coleoptile was only slightly more resistant to ethanol than the other tissues in short term experiments. However, the rice coleoptile in anoxia retained substantial ability to absorb both glucose and sucrose, and leakage of solutes was much less than that observed with the other tissues.
Article
Intact protoplasts are ruptured by rapid centrifugation through a narrow-aperture nylon mesh and the intact chloroplasts are then separated from the cytoplasm by sedimentation through a layer of silicone oil below the mesh. Within 6 to 8 s of starting the centrifuge, 90% of the chloroplasts are separated into the pellet fraction which contains only 10 to 15% contamination by mitochondria and peroxisomes and less than 5% contamination by soluble cytoplasm as judged by the distribution of marker enzymes. This technique should allow determination of the distribution of metabolites between the chloroplast and cytoplasmic compartments of intact protoplasts.
Article
Spinach protoplasts are useful in lipid metabolism studies because they provide a homogeneous, differentiated plant tissue that can take up precursors directly from the medium. they are the material of choice for subcellular localization because they can be easily lysed and the intact organelles separated on density gradients. Crude fungal enzyme preparations are used to degrade the cell wall of the spinach leaves, which are stripped of their epidermal cells by brushing and sliced finely to increase the surface for enzyme action. Protoplasts are harvested from the digested leaf slices by filtration and washed and purified by repeated centrifugation. If protoplasts are to be used in metabolism studies, they are stabilized with a salt solution containing divalent cations and incubated in strong light with lipid or fatty acid precursors. For subcellular localization studies, salts are not present in the wash medium to reduce chloroplast aggregation in the gradients and protoplasts are lysed by shearing in a microemulsifying needle or passage through 20- and 10-μm mesh before sucrose density gradient centrifugation. Comparison with marker enzymes for organelles provides a method for the complete subcellular localization of an enzyme activity.
Article
The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase, sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO(2) occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development.
Article
A method was developed for the large scale and rapid isolation of intact viable corn root protoplasts. Pure and metabolically active protoplasts were collected using a flotation technique. Vital staining tests, light and electron microscopy, and measurements of basic metabolic processes indicated that the isolated protoplasts were metabolically active, and that the plasmalemma and other organelles were well preserved. The isolated protoplasts performed normal, active ion transport functions. Time course of K(+) and inorganic phosphate (H(2)PO(4) (-)) influx and the effects of external pH, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, fusicoccin, and diethylstilbestrol on K(+) and inorganic phosphate influx and net H(+) efflux in isolated protoplasts correlated well with data obtained on root segments. Data presented indicated that isolated protoplasts from roots can be used to gain additional insights into the mechanism of ion transport in plant cells.
Article
Subcellular organelles from castor bean (Ricinus communis) endosperm were isolated on discontinuous sucrose gradients from germinating seeds which were 1 to 7 days postimbibition. Marker enzyme activities of the organelles were measured (fumarase, catalase, and triose phosphate isomerase) and the homogeneity of the organelle fractions was examined by electron microscopy. Pyruvate dehydrogenase complex activity was measured only in the mitochondrial fraction and attempts to activate or release the enzyme from the proplastid were not successful. A pathway is proposed for the most efficient use of endosperm carbon for de novo fatty acid biosynthesis that does not require the presence of the pyruvate dehydrogenase complex in the proplastid to provide acetyl-coenzymeA.
Article
This chapter discusses the preparation of protoplasts from the plant tissues for organelle isolation. Isolated protoplasts offer some advantages as an experimental system for the studies of plant cell biology, one of which is used as a starting material for the preparation of constituent organelles. The chapter discusses methods for preparing protoplasts from three different plant tissues. Several dye staining techniques and some cellular activities, such as respiration and photosynthesis have been employed as markers of potential activities of isolated protoplasts. However, these methods do not provide a quantitative measure of intactness, and do not secure the intrinsic potential activities of the original cell. Protoplasts have been isolated from oat and barley aleurone layers, but only the oat protoplasts were shown to produce aamylase in response to gibberellin A3 (GA3). The preparation of intact protoplasts from mature barley aleurone layers, capable of producing GA3-responsive α-amylase, has been presented in the chapter. A simple and rapid method was developed for the quantitative analysis of intactness of isolated protoplasts using glycolate oxidase activity as an index. This method is generally applicable to green leaf tissues, and in fact, has been applied to grapevine protoplasts.
Article
ATP citrate lyase (EC 4.1.3.8) has been found in crude extracts from endosperm tissue of germinating castor bean and shows its maximum activity in 4- to 5-day-old seedlings. A strict requirement for coenzyme A and adenosine 5'-triphosphate was demonstrated. The pH optimum for the reaction is around 7.5. The unstable enzyme can be stabilized by freezing and addition of citrate and glycerol. (-)-Hydroxycitrate is a potent inhibitor. The molecular weight is about 400,000. The adenosine 5'-triphosphate citrate lyase is localized in the plastids, where it possibly plays a role in providing acetyl coenzyme A for lipid biosynthesis.
Article
Intact mitochondria were prepared from spinach (Spinacia oleracea L. var. Kyoho) leaf protoplasts and purified by Percoll discontinuous gradient centrifugation. Assays of several marker enzymes showed that the final mitochondrial preparations obtained are nearly free from other contaminating organelles, e.g. chloroplasts, peroxisomes, and endoplasmic reticulum. These mitochondria oxidized malate, glycine, succinate, and NADH, tightly coupled to oxidative phosphorylation with high values of ADP to O ratio as well as respiratory control ratio. The rate of NADH oxidation was 331 nmoles O(2) per milligram mitochondrial protein per minute, which is comparable to that obtained by highly purified potato or mung bean mitochondria. However, the activity of glutamine synthetase was barely detectable in the isolated mitochondrial fraction. This finding rules out a hypothetical scheme (Jackson, Dench, Morris, Lui, Hall, Moore 1971 Biochem Soc Trans 7: 1122) dealing with the role of the mitochondrial glutamine synthetase in the reassimilation of NH(3), which is released during the step of photorespiratory glycine decarboxylation in green leaf tissues, but it is consistent with the photosynthetic nitrogen cycle (Keys, Bird, Cornelius, Lea, Wallsgrove, Miflin 1978 Nature (Lond) 275: 741), in which NH(3) reassimilation occurs outside the mitochondria.
Article
Two isoenzymes each of phosphoglucomutase, hexose phosphate isomerase, aldolase, fructose diphosphatase, phosphofructokinase, and 6-phosphogluconate dehydrogenase have been separated by DEAE-cellulose column chromatography of extracts from endosperm of germinating castor beans (Ricinus communis cv. Hale). One of each of the enzymes is localized in the cytosol and the other is confined to plastids. Developmental studies of these isoenzymes were carried out to clarify their roles in the endosperm. In extracts from ungerminated seeds the activities of marker enzymes of mitochondria (fumarase), plastids (ribulose bisphosphate carboxylase), and glyoxysomes (catalase) were low, but phosphoglucomutase, hexose phosphate isomerase, aldolase, and 6-phosphogluconate dehydrogenase were present in relatively high activity. The total amounts of these enzymes increased 3- to 4-fold during the first 5 days of growth. The activities of isoenzymes in the plastids rose in parallel with that of ribulose bisphosphate carboxylase to reach a maximum at day 4, and like the carboxylase they declined sharply thereafter. The activities of the cytosolic isoenzymes peaked at day 5. These changes are consistent with the roles previously proposed for the sequences present in plastid and cytosol.
Article
Isolated cauliflower (Brassica oleracea) bud plastids, purified by isopycnic centrifugation in density gradients of Percoll, were found to be highly intact, to be practically devoid of extraplastidial contaminations, and to retain all the enzymes involved in fatty acid, phosphatidic acid, and monogalactosyldiacylglycerol synthesis. Purified plastids possess all the enzymes needed to convert triose phosphate to starch and vice versa, and are capable of conversion of glycerate 3-phosphate to pyruvate for fatty acid synthesis. They are also capable of oxidation of hexose phosphate and conversion to triose phosphate via the oxidative pentosephosphate pathway. Cauliflower bud plastids prove to be, therefore, biochemically very flexible organelles.
Article
A method was developed for the quantitative analysis of intactness of spinach leaf protoplasts using glycolate oxidase activity as an index. Since glycolate does not penetrate into protoplasts at neutral pH, the increase of O(2) consumption by the addition of glycolate to protoplast suspension was due to the glycolate oxidase activity released from damaged protoplasts. The proportion of damaged protoplasts in the whole preparation was calculated from the ratio of released and total glycolate oxidase activity. Freshly prepared spinach leaf protoplasts were found to be 80 to 90% intact as estimated by the method. The effect of osmolarity on the respiratory activities of spinach leaf protoplasts was also examined by applying the same principle.
Article
Membranes were isolated from protoplasts of Rubus hispidus cells cultured in vitro and then separated with sucrose and Dextran T-70 gradients. Two peaks of ATPase activity were obtained. The first peak and proton pumping activity (density 1.085) was inhibited by both vanadate and nitrate. The second peak (density 1.150), was also inhibited by vanadate but not by nitrate; it closely coincided with UDP glucose-sterol-β-D-glucosyltransferase activity, a marker for plasma membrane. Tonoplast fractions isolated from vacuoles were characterized by the same nitrate- and vanadate-sensitive H+ translocating ATPase as described for the gradients.
Article
This chapter describes a method for mechanical blending of the tissue and purification of plastids on a self-generating isosmolar density gradient of Percoll. The method described produces plastids containing middle-size starch grains with a high degree of intactness. They approach ultimate purity as judged by electron microscopy and analysis using marker enzymes. Percoll-purified cauliflower bud plastids are thus suitable for a large range of studies. For example, the first exhaustive description of the enzymatic capacities of a nongreen plastid type for basic carbon metabolism (glycolysis, pentose-P pathway, and starch metabolism), nitrite assimilation, and lipid synthesis (long-chain fatty acid and galactolipid synthesis) can be drawn up by using purified cauliflower bud plastids. The maximal activities recorded for the enzymes involved in these pathways showed that cauliflower bud plastids are recovered in an excellent physiological state. Cauliflower bud plastids are also suitable for envelope phospholipid and pigment analysis, native plastid-DNA isolation, metabolite transport studies, and helps to increase the knowledge about these biochemically very flexible organelles.
Article
This chapter describes a method for the isolation of amyloplasts from developing endosperm of maize. Probably the most critical stage in the isolation of intact amyloplasts from maize endosperm is the release of the amyloplasts from the cells. Amyloplasts containing a single large starch granule, such as potato and maize, is very prone to rupture during even low-gravity centrifugation. Although castor bean endosperm protoplasts were ruptured with little damage to plastids by passage through nylon mesh, the presence of large starch granules in the maize amyloplasts creates a special problem. For example, during maize endosperm protoplast rupture the starch granules quickly settle and cake on the surface of the nylon mesh. The trypsin treatment is shown to increase the rate of maize amyloplast membrane rupture and thus resulted in a low estimate of percentage intactness. Dihydroxyacetone phosphate (DHAP) readily crosses the maize amyloplast membrane and is converted to starch.
Article
Guard cell and mesophyll cell protoplasts of Vicia faba L. were purified and separated into cytoplasmic and plastid fractions by a selective silicone-oil filtration. Before fractionation, the protoplasts were ruptured by a low speed centrifugation through a narrow-aperture nylon net placed in a plastic vial. This protoplast homogenation and subsequently the silicone-oil fractionation offer the possibility of investigating the comparatmentation of the enzymatic carboxylating (ribulose bisphosphate carboxylase EC 4.1.1.39, phosphoenolpyruvate carboxylase EC 4.1.1.31, NAD(+) and NADP(+) linked malate dehydrogenase EC 1.1.1.37) and decarboxylating pathways of malic (malic enzyme EC 1.1.1.40, phosphoenolpyruvate carboxykinase EC 4.1.1.32, pyruvate orthophosphate dikinase EC 2.7.9.1) which occur during the swelling and shrinking of the guard cell protoplasts. A model is proposed which describes the transport processes of malic acid during the starch-malate balance as correlated to the volume changes of the protoplasts. As the enzymes and their compartmentation in the guard cell protoplasts seem to be consistent with those of crassulacean acid metabolism (CAM) plants, the metabolism of stomata and of CAM cells is compared.
Chapter
Soon after Nagata and Takebe [1] showed that isolated tobacco leaf mesophyll protoplasts have the capability to regenerate cell walls and divide in vitro, Sakai and Takebe [2] reported that these protoplasts incorporated precursors into acid-insoluble fractions of RNA and protein. Several subsequent studies were aimed at investigating further the capacity of isolated protoplasts to synthesize RNA and protein. Thus Wasilewska and Kleczkowski [3] used protoplasts from maize seedling shoot tips to follow RNA synthesis, and reported that gibberellic acid promoted this synthesis and increased the poly (A)+ RNA fraction. As their protoplasts did not show cell division, the incorporation of uridine was terminated after 2–3 hours. Using cotyledon and leaf cucumber protoplasts, Coutts et al. [4] reported that uridine was incorporated into ribosomal RNA and that this incorporation was rather low at first but increased gradually up to 1 or 2 days. Such an increase of precursor incorporation was also indicated for tobacco mesophyll protoplasts [5]. Further examination of this and other protoplast systems indicated that the density of the protoplast suspension during culture strongly affects RNA and protein precursor incorporation; that lectins improve leucine, uridine, and thymidine incorporation; and that although the incorporation of these precursors increases steadily during early culture, it is strongly reduced by osmotic stress.
Chapter
Compartmentation of plant constituents is an important means of regulating plant metabolism (Dennis and Miernyk 1982). Perhaps the most significant breakthrough in the last 10 years in this field has been the use of protoplasts as a source for cell fractionation experiments and organelle isolations. Although protoplasts were first isolated using cell-wall-degrading enzymes in 1960 (Cocking 1960), it was not until the mid 1970’s that functional organelles were isolated from protoplasts (Galun 1981; Nishimura et al. 1976; Rathnam and Edwards 1976; Wagner and Siegelman 1975). Since that time, protoplasts have been used in hundreds of studies of plant metabolism.
Chapter
Biochemical studies on leaf proteins carried out by Wildman and Bonner (1947) revealed the presence of a major protein component having a large molecular size (18s) which was designated as fraction-1-protein. The ubiquitous distribution of this protein in green plant leaves and green algae, as determined by analytical ultracentrifugation and immunological precipitation methods, stimulated later studies on its enzymic nature (Dorner et al., 1958). The independent investigation of the path of carbon in photosynthetic CO2 fixation, together with that on the enzymic machinery of the reductive pentose phosphate cycle, led to the discovery of ribulose-1,5-bisphosphate (RuBP) carboxylase (E.C. 4.1.1.39; carboxydismutase) catalyzing the following key reaction [Eq. (1); Quayle et al., 1954; Weissbach et al., 1954, 1956; Chap. II.1, this vol.] $$ {\rm{RuBP + C}}{{\rm{O}}_2}{\rm{ }}{\rm{ 2}}\left( {{\rm{PGA}}} \right) $$ (1)
Article
Purified intact protoplasts were isolated from etiolated and greening leaves of Avena sativa. They were ruptured by forcing them through a 20-μm aperture nylon net and immediately thereafter fractionated into a pure pellet of plastids (well above 70% of total plastids), a layer of mitochondria only slightly contaminated by other cellular constituents (about 50% of total mitochondria), and a cytoplasmic supernatant. This was achieved within 60 s by an integrated method of homogenation of protoplasts and centrifugal filtration of the homogenate on a gradient of silicone oils, contained together with the nylon net in 450 μl microtubes, and verified by comparing the levels of activity of specific markers within the three fractions obtained. With appropriate modifications to immediately quench metabolic reactions within the fractions, this method allows the determination of metabolite levels within plastids, mitochondria, and the cytoplasmic compartment of intact protoplasts. The applicability of this technique is demonstrated by the determination of ATP in the plastids, mitochondria, and the cytoplasm of protoplasts obtained from etiolated and greening primary leaves of Avena. The levels of ATP, corrected for contamination of the fractions by each other, exhibit a pronounced transient increase during greening, especially within the cytoplasm.
Article
A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,L-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm(-3). Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm(-3). Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each.
Article
The cellular distribution of enzymes involved in nitrogen assimilation: nitrate reductase (EC 1.6.6.2), nitrite reductase (EC 1.6.6.4), glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 2.6.1.53), and glutamate dehydrogenase (EC 1.4.1.3) has been studied in the roots of five plants: maize (Zea mays L. hybrid W 64A x W 182E), rice (Oryza sativa L. cv. Delta), bean (Phaseolus vulgaris L. cv. Contender), pea (Pisum sativum L. cv. Demi-nain), and barley (Hordeum vulgare L.). Initially, cell organelles were separated from soluble proteins by differential centrifugation. Cell organelles were also subjected to sucrose density gradients. The results obtained by these two methods indicate that nitrite reductase and glutamate synthase are localized in plastids, nitrate reductase and glutamine synthetase are present in the cytosol, and glutamate dehydrogenase is a mitochondrial enzyme.
Article
The process of carrot (Daucus carota L.) somatic embryogenesis is highly sensitive to exogenously added ethanol, since 5 mM ethanol inhibits this process by 50%, whereas the growth of proliferating carrot cells is inhibited to the same extent by 20 mM ethanol. This is consistent with the fact that proliferating cultures produce ethanol and release it into the medium at concentrations up to 20 mM, whereas embryogenic culture medium contains less than 1 mM ethanol. Data are presented showing the influence of cell density and 2,4-dichlorophenoxyacetic acid on ethanol production and on the presence of an alcohol-dehydrogenase (EC 1.1.1.1.) inactivator in carrot embryos.
Article
Plastid and mitochondrial DNAs from suspension cultures of Daucus carota subsp. sativus cv. Danvers, D. carota subsp. gummifer, D. capillifolius, and D. pusillus were compared by restriction endonuclease fragment analysis. Organelles isolated from protoplasts of suspension cultures were purified using a one-step sucrose gradient. Plastid DNA fragment patterns for subsp. sativus, subsp. gummifer, and capillifolius were indistinguishable in Pstl, SalI and XhoI enzyme digests. No variation was detected in BamHI or HindIII digests between subsp. sativus and capillifolius. Pusillus plastid DNA varied significantly when compared to the other Daucus cultures. The size of the plastid genome of each species as determined by fragment addition, was estimated at 155 kb. Restriction digests of the mitochondrial DNAs generated a large number of fragments which when totaled established a size of 386–468 kb for the genomes. Densitometer scans of the fragment patterns indicate the bands were present in variable stoichiometry. Up to 26% of the fragments generated by PstI, SalI and HindIII digests were homologous in size among the four mitochondrial genomes. A further comparison of mitochondrial fragments indicated a closer homology of subsp. sativus to capillifolius than to subsp. gummifer or pusillus as was also found with the plastid genomes.
Article
A method for obtaining protoplasts from highly cyanogenic endosperm tissue of Hevea brasiliensis has been developed. The isolated protoplasts were characterized. Intracellular as well as extracellular occurrence of the Hevea linamarase ((3-glucosidase) and a-mannosidase was demonstrated by comparing the enzyme activities of homogenates of endosperm tissue with those of isolated protoplasts and confirmed by the finding that these enzyme activities are present in high percentages in rinsing solutions of intact endosperm tissue.
Article
Conditions have been systematically evaluated for the isolation of viable protoplasts from endosperm tissue of developing wheat grain (Triticum aestivum L. cv. Mardler). Optimal conditions are as follows: an enzyme-mixture of 1% pectinase and 2% cellulase in 0.3 M mannitol and 0.2 M sucrose as osmoticum at pH 5.5; 2 h at 28°C or 16 h at 4°C incubation; 1 × g sedimentation for protoplast purification; centrifugation and abrupt changes in pH or temperature should be avoided to prevent protoplast lysis. A yield of 1–5 × 105 endosperm protoplasts per gram fresh weight of grain was obtained with this procedure. This was estimated to represent 10–50% of the original endosperm cells. Approximately 80% of the protoplasts excluded Evans blue dye. Enzyme latency experiments measuring uridine diphosphate glucose (UDPG) pyrophosphorylase activity showed that more than 80% of the protoplasts were intact. The protoplasts were able to incorporate radiolabelled sucrose or glucose into starch at rates comparable to isolated endosperm tissues. These data indicate that the protoplasts are metabolically active and sufficiently free from contamination to permit further metabolic studies and the development of plastid isolation procedures.
Article
Summary To identify markers for fusion and transformation studies, cell suspension cultures of four members of theDaucus genus were examined to determine differences in culture conditions, isoenzyme patterns, and plastid DNA. The four were:D. carota subsp.sativus cv. Danvers,D. carota subsp.gummifer, D. capillifolius, andD. pusillus. Under appropriate conditions, all four grew well as liquid cell suspension cultures and regenerated from protoplasts into plants. Enzyme activities of homoserine dehydrogenase (HSDH) and alcohol dehydrogenase from cell culture extracts were analyzed on electrophoretic gels. Although only one form of HSDH was present in eachDaucus line, the rate of migration of HSDH from cv. Danvers was different from that of the other cell lines. Multiple isoenzymic forms of ADH were present in eachDaucus cultivar. Camparison of endonuclease restriction fragment patterns from plastid DNAs digested by BamHI revealed only small differences between plastid DNAs of cv. Danvers and subsp.Gummifer, whereas large differences were observed between cv. Danvers andD. pusillus plastid DNA patterns. No differences were found between cv. Danvers andD. capillifolius plastid DNA patterns when examined using eight different restriction enzymes. The data indicate that specific isoenzyme and organelle DNA restriction fragment patterns will be useful markers for precise identification of genomes of differentDaucus species in somatic hybridization experiments.
Article
Intact mitochondria wereprepared fromspinach (Spinacia oeracea L. var. Kyoho) leaf protoplasts andpurified byPercoll discontinuous gradient centrifugation. Assays ofseveral marker enzymes showed thatthefinal mitochondrial preparations obtained arenearly free fromother contami- nating organelles, e.g. chloroplasts, peroxisomes, andendoplasmic reticu- lum.These mitochondria oxidized malate, glycine, succinate, andNADH, tightly coupled tooxidative phosphorylation withhigh values ofADPto0 ratio aswell asrespiratory control ratio. TherateofNADHoxidation was 331nmoles 02permilligram mitochondrial protein perminute, which is comparable tothatobtained byhighly purified potato ormungbean mitochondria. However, theactivity ofglutamine synthetase wasbarely detectable intheisolated mitochondrial fraction. Thisfmding rules outa hypothetical scheme(Jackson, Dench, Morris, Lui, Hall, Moore1971 Biochem SocTrans7:1122) dealing withtherole ofthemitochondrial glutamine synthetase inthereassimilation ofNH3,which isreleased during thestepofphotorespiratory glycine decarboxylation ingreen leaf tissues, butitisconsistent withthephotosynthetic nitrogen cycle (Keys, Bird, Cornelius, Lea,Wallsgrove, Miffin 1978Nature (Lond) 275: 741), inwhich NH3reassimilation occurs outside themitochondria.
Article
ABSTRACr A method wasdeveloped forthequantitative analysis ofintactness of spinach leaf protoplasts using glycolate oxidase activity asanindex. Since glycolate doesnotpenetrate into protoplasts atneutral pH,the increase of02consumption bytheaddition ofglycolate toprotoplast suspension wasduetotheglycolate oxidase activity released fromdam- agedprotoplasts. Theproportion ofdamaged protoplasts inthewhole preparation wascalculated fromtheratio ofreleased andtotal glycolate oxidase activity. Freshly prepared spinach leaf protoplasts werefound to be80to90%intact asestimated bythemethod. Theeffect ofosmolarity ontherespiratory activities ofspinach leaf protoplasts wasalso examined byapplying thesameprinciple.
Article
Full-text available
SUMMARY Protoplasts isolated from suspension-cultured tobacco cells were inoculated with tobacco mosaic virus (TMV) using a procedure similar to that for tobacco mesophyll protoplasts. Fifty to 70~ of protoplasts were infected, as determined by staining with fluorescent TMV antibody. The formation of progeny virus particles was substantiated by electron microscopy of sectioned protoplasts, and the virus yield was assessed by assaying the infectivity of protoplast extracts. The course of TMV multiplication was studied by following the incorporation of (3H)uridine into virus RNA. The results indicated that infection in this system is synchronous and that the rate of virus multiplication is comparable to that in mesophyll protoplasts. A system of undifferentiated, growing plant cells was thus established for one-step growth of TMV.
Article
Methods for the formation of protoplasts from developing maize endosperm and for the aqueous isolation of intact amyloplasts from such protoplasts are described. Protoplasts were obtained after incubating endosperm slices in a medium containing cellulase and pectolyase for 5 days at 4 degrees C or 5 hours at 30 degrees C. After purification in a Ficoll density gradient, the protoplasts were reptured by forcing the suspension through a Nitex mesh (20 micrometer) positioned at the lower end of a modified disposable syringe. The resulting filtrate was layered on a discontinuous Ficoll density gradient of 30, 15, and 10%. Each Ficoll solution contained 0.7 molar sucrose, 10 millimolar arginine, 10 millimolar dl-dithiothreitol, 50 millimolar 2-(N-morpholino)ethanesulfonic acid (pH 5.6), and 2 millimolar CaCl(2). After 3 hours in the cold, an amyloplast fraction 50 to 93% intact and free from cytoplasmic, mitochondrial, and glyoxysomal contamination was recovered in the 15% Ficoll layer. Amyloplast intactness was estimated by fluorescent microscopy and activity of certain amyloplast marker enzymes before and after rupture of the amyloplast membrane. Starch branching enzyme, ADPG-pyrophosphorylase, and nitrite reductase were used as amyloplast marker enzymes.
Article
Intact plant vacuoles were prepared in large numbers (106) from protoplasts of mature leaves, flower petals, stems, pedicels, filaments, styles, and young fruits. Treatment of protoplasts with 0.2 molar K2HPO4-HCl, pH 8, with slow stirring resulted in gentle osmotic rupture of the protoplasts and release of intact vacuoles. Particulate components of the protoplast, less the vacuole, were largely shed as an aggregate, which was removed by filtration. Vacuoles were recovered from the filtrate by low-speed centrifugation. The general procedure was also used to isolate chloroplasts with a high degree of integrity and excellent photochemical activity.
Article
A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1-14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.
Article
The occurrence and subcellular distribution of enzymes of the cytidine diphosphate choline pathway of lecithin synthesis have been examined. Choline kinase (EC 2.7.1.32) was completely soluble, while phosphorylcholine-cytidyl transferase (EC 2.7.7.15) and phosphorylcholine-glyceride transferase (EC 2.7.8.2) were associated with particulate fractions. Although components sedimenting at 10,000 to 100,000 x g contained both enzymes, phosphorylcholine-cytidyl transferase and particularly phosphorylcholine-glyceride transferase were present in the 10,000 x g pellet, which contained the major organelles, mitochondria, and glyoxysomes. When the crude homogenate was centrifuged on a sucrose density gradient, four major bands of particulate protein were recovered. A band at density 1.24 g/cm(3) contained the glyoxysomes and was devoid of phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase activity. Enzyme activity was barely detectable in the mitochondria, at density 1.18 g/cm(2). Phosphorylcholine-glyceride transferase was found almost exclusively in a sharp band at density 1.12 g/cm(3), and phosphorylcholinecytidyl transferase was found in the uppermost band at density 1.08 g/cm(3). Thus, for the synthesis of lecithin in their membranes, the glyoxysomes and mitochondria depend on enzymes elsewhere in the cell; the final two steps in lecithin formation occur, apparently exclusively, in separate particulate cell components.
Article
Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm(3) which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50-60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [(14)C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21-1.22 g/cm(3), whereas the original glyoxysomes appeared at density 1.24 g/cm(3). Electron microscopy showed that the fraction at 1.21-1.22 g/cm(3) was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.
Article
The fatty layer obtained after centrifuging a macerate of ungerminated castor beans, Ricinus communis, contains an active lipase. The fatty layer has been examined by a combination of biochemical and electron microscopic techniques before and after periods of active lipolysis with Pb++ in the reaction mixture. The results confirm the presence of spherosomes, the fat storage organelles of the cells, concentrated in the fatty layer. Lipase activity is localized in the spherosomes derived from endosperm tissue of ungerminated seeds.
Article
A spectrophotometric method of measuring the enzymatic formation and disappearance of umaric and cis-aconitic acids is reported.RésuméNous décrivons une methode spectrophotométrique qui permet de mesurer la formation et la disparation enzymatique de l'acide fumarique et de l'acide cis-aconitique.ZusammenfassungEine spektrophotometrische Methode zur Messung der enzymatischen Bildung und Zerstörung von Fumarsäure und cis-Akonitsäure wird beschrieben.
Article
A substantial portion of the ribulose 1,5-diphosphate carboxylase activity in the endosperm of germinating castor beans (Ricinus communis var. Hale) is recovered in the proplastid fraction. The partially purified enzyme shows homology with the enzyme from spinach (Spinacia oleracea) leaves, as evidenced by its reaction against antibodies to the native spinach enzyme and to its catalytic subunit. The enzyme from the endosperm of castor beans has a molecular weight of about 500,000 and, with the exception of a higher affinity for ribulose 1,5-diphosphate, has similar kinetic properties to the spinach enzyme. The castor bean carboxylase is inhibited by oxygen and also displays ribulose 1,5-diphosphate oxygenase activity with an optimum at pH 7.5.
Article
Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal "ghosts" at equilibrium density 1.21 g/cm(3) on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.
Article
A technique for the isolation of intact plastids from spinach (Spinacia oleracea) and pea (Pisum sativum) leaves, pea roots and castor bean (Ricinus communis) endosperm is described. This technique involves brief centrifugation of whole homogenates on density gradients. Intact plastids were located in the gradient by assaying for triose phosphate isomerase activity. Contamination of the plastic peak with mitochondria and microbodies was estimated by measurement of cytochrome oxidase and catalase, respectively. For three of the four tissues the level of contamination of the plastids by these organelles was 2% or less. The sedimentation behavior of microbodies from different tissues is discussed.
Article
A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA.
Article
Glyoxysome, endoplasmic reticulum, mitochondria, and proplastid fractions were isolated from endosperm of castor beans (Ricinus communis) germinated for 5 days at 30 C. Samples from sucrose density gradients were diluted with 0.15 m KCI and the membranes pelleted. Lipid extracts of these membranes were analyzed for phosphoglyceride, acyl lipid, and sterol content. The endoplasmic reticulum contains 1.24 mumol of phosphoglyceride per mg of protein; the mitochondria, 0.65 mumol/mg; and the glyoxysome membranes, 0.55 mumol/mg. Phosphatidyl choline and phosphatidyl ethanolamine are the most abundant lipids in all membranes studied, accounting for 70% or more of the lipid phosphorus and 50% or more of the fatty acid. Glyoxysome membranes and endoplasmic reticulum also contain phosphatidyl inositol (respectively, 9 and 17% of the lipid phosphorus) and free fatty acids (13% of the total fatty acid in each). Compared with other organelles, mitochondrial membranes have more phosphatidyl ethanolamine relative to phosphatidyl choline and are characterized by the presence of cardiolipin, in which 80% of the fatty acid is linoleate. The relative amounts of linoleate, palmitate, oleate, stearate, and linolenate in each of the phosphotoglycerides are constant regardless of the membrane source. Stimasgasterol and beta-sitosterol are present in the membranes (1-9 nmol each/mg protein).The data provide further evidence that glyoxysome membranes are derived from the endoplasmic reticulum but at the same time indicate some differentiation.
Article
Freshly prepared spinach leaf protoplasts were gently ruptured by mechanical shearing followed by sucrose density gradient centrifugation to separate constituent cell organelles. The isolation of intact Class I chloroplasts (d = 1.21) in high yield, well separated from peroxisomes and mitochondria, was evidenced by the specific localization of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), NADP triose-P dehydrogenase (EC 1.2.1.9), and carbonic anhydrase (EC 4.2.1.1) in the fractions. A clear separation of chloroplastic ribosomes from the soluble cytoplasmic ribosomes was also demonstrated by the band patterns of constituent RNA species in the polyacrylamide gel electrophoresis. Localization of several enzyme activities specific to leaf peroxisomes, e.g. catalase (EC 1.11.1.6), glycolate oxidase (EC 1.1.3.1), glyoxylate reductase (EC 1.1.1.26), glutamate glyoxylate aminotransferase (EC 2.6.1.4), serine glyoxylate aminotransferase, and alanine glyoxylate aminotransferase (EC 2.6.1.12) in the peroxisomal fractions (d = 1.25), was demonstrated. Overall results show the feasibility of the method for the isolation of pure organelle components in leaf tissues.
Article
The intracellular distribution of enzymes capable of catalyzing the reactions from oxaloacetate to sucrose in germinating castor bean endosperm has been studied by sucrose density gradient centrifugation. One set of glycolytic enzyme activities was detected in the plastids and another in the cytosol. The percentages of their activities in the plastids were less than 10% of total activities except for aldolase and fructose diphosphatase. The activities of several of the enzymes present in the plastids seem to be too low to account for the in vivo rate of gluconeogenesis whereas those in the cytosol are quite adequate. Furthermore, phosphoenolypyruvate carboxykinase, sucrose phosphate synthetase, and sucrose synthetase, which catalyze the first and final steps in the conversion of oxaloacetate to sucrose, were found only in the cytosol. It is deduced that in germinating castor bean endosperm the complete conversion of oxaloacetate to sucrose and CO(2) occurs in the cytosol. The plastids contain some enzymes of the pentose phosphate pathway, pyruvate dehydrogenase and fatty acid synthetase in addition to the set of glycolytic enzymes. This suggests that the role of the plastid in the endosperm of germinating castor bean is the production of fatty acids from sugar phosphates, as it is known to be in the endosperm during seed development.
Localization of enzymes within microbodies 4. KING PJ, HE STREET 1973 Growth pattern in cell cultures
  • Huang Ahc
HUANG AHC, H BEEVERS 1973 Localization of enzymes within microbodies. J Cell Biol 58: 379-389 4. KING PJ, HE STREET 1973 Growth pattern in cell cultures. In HE Street, ed, Plant and Cell Tissue Culture. University of California Press, Berkeley, pp 269-337
Distribution of gluconeogenic enzymes in the castor bean endosperm
  • Mj Kobr
  • H Beevers
KoBR MJ, H BEEVERS 1968 Distribution of gluconeogenic enzymes in the castor bean endosperm. Plant Physiol 43: S-17
Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm 7. LUCK H 1965 Catalase Methods of Enzymatic Analysis Isolation of intact plastids from a range of plant tissues
  • T Kagawa
LORD M, T KAGAWA, H BEEVERS 1972 Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm. Proc Nat Acad Sci USA 69: 2429-2432 7. LUCK H 1965 Catalase. In HU Bergmeyer, ed, Methods of Enzymatic Analysis. Academic Press, New York, pp 885-894 8. MIFFLIN BJ, H BEEVERS 1974 Isolation of intact plastids from a range of plant tissues. Plant Physiol 54: 870-874
A rapid, simple method for nuclei isolation from plant protoplasts Association of lipase activity with spherosomes of Ricinus communis Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm
  • Le Relcher
  • Ly Yatsu
  • Osmond Cb
  • T Akazawa
OHYAMA K, LE RELCHER, D HORN 1977 A rapid, simple method for nuclei isolation from plant protoplasts. Plant Physiol 60: 179-181 13. ORY RL, LY YATSU, HW KIRCHER 1968 Association of lipase activity with spherosomes of Ricinus communis. Arch Biochem Biophys 264: 255-264 14. OSMOND CB, T AKAZAWA, H BEEVERS 1975 Localization and properties of ribulose diphosphate carboxylase from castor bean endosperm. Plant Physiol 55: 226-230
Assay methods for the key enzymes of the glyoxylate cycle
  • Dixon Gh
  • Hl Kornberg
DIXON GH, HL KORNBERG 1959 Assay methods for the key enzymes of the glyoxylate cycle.
Enzymatic changes during the differentiation of tissues in young pea roots
  • Sutcliffe Jf
  • R Sexton
SUTCLIFFE JF, R SEXTON 1974 Enzymatic changes during the differentiation of tissues in young pea roots. In J Kolek, ed, Structure and Function of Primary Root Tissues. Veda, Bratislava, pp 203-219
Fatty acid synthesis in germinating castor bean endosperm
  • H Beevers
VICK B, H BEEVERS 1977 Fatty acid synthesis in germinating castor bean endosperm. Plant Physiol 59: S-31
Growth pattern in cell cultures Plant and Cell Tissue Culture
  • King Pj
  • He Street
KING PJ, HE STREET 1973 Growth pattern in cell cultures. In HE Street, ed, Plant and Cell Tissue Culture. University of California Press, Berkeley, pp 269-337
Spectrophotometric measurements of enzymatic formation of fumaric and cisaconitic acids
RACKER E 1950 Spectrophotometric measurements of enzymatic formation of fumaric and cisaconitic acids. Biochim Biophys Acta 4: 211-214