Polypeptide pattern and enzymic character of vacuoles isolated from barley mesophyll protoplasts

Intact chloroplasts and vacuoles were isolated from mesophyll protoplasts of barley. The chloroplasts occupied about 15% of the cellular volume and contained 75% of the protein, whereas the vacuoles occupied about 80% of the volume and contained less than 4% of total cellular protein. Contamination of the vacuolar fraction by foreign protein is included in these values. Chlorophyll was absent from the vacuolar fraction, but less than 1% of several extra-vacuolar marker proteins were still present. The vacuoles contained hydrolytic enzymes. Several of them (-mannosidase, -galactosidase, N-acetylglucosaminidase) were soluble, whereas part of the activity of others semimented with the tonoplasts during centrifugation. Attached proteins could be released from the membranes during freezing in the presence of NaCl. One-dimensional gel electrophoretic separation of soluble vacuolar proteins under non-denaturing conditions yielded more than 10 protein bands. A comparative analysis was performed of thylakoids and vacuoles which were subfractionated into tonoplasts and soluble vacuolar constituents. Sodium dodecyl sulfate gel electrophoresis separated about 15 polypeptides of the soluble fraction which reacted with silver reagent. The tonoplast fraction yielded about 20 bands. A similar number of bands was observed when vacuoles incubated with the ¹⁴C-labelled SH-reagent N-ethylmaleimide were analysed for radioactive polypeptides. Silverstaining of the polypeptides and their SH-content did not correlate. Several polypeptides of the vacuolar fraction had molecular weights very similar to the molecular weights of known chloroplast proteins. However, with the exception of the two subunits of ribulose-1,5-bisphosphate carboxylase, contamination of the vacuolar fraction by chloroplast proteins could be ruled out as a possible cause of the close correspondence. The lipophilic carboxylic-group reagent N,N-dicyclohexylcarbodiimide ([¹⁴C]DCCD) reacted with several polypeptides of thylakoids and tonoplasts. However, the labelling patterns were different. The most heavily labelled polypeptide of thylakoids was the 8-kDa polypeptide of the basal part of the coupling factor CF₀. Tonoplast polypeptides heavily labelled with [¹⁴C]DCCD had molecular weights of 24, 28, and 56 kDa. The vacuolar 8-kDa polypeptide remained unlabelled.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - IA iodoacetamide - NEM N-ethylmaleimide - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate     

Studies on vacuole regeneration in evacuolated tobacco mesophyll protoplasts. Protein analysis by one- and two-dimensional microgel-electrophoresis

Evacuolated protoplasts, P(–), have been prepared from mesophyll protoplasts. P(+), of Nicotiana tabacum L. (cv. Samsun) by centrifugation in an iso-osmotic Percoil gradient. In comparative analyses performed with these two types of cells, vacuole proteins should appear in P(+) extracts, but be absent from freshly isolated P(–). Such differences were detectable in total protein extracts after two-dimensional electrophoresis. Four spots were located that are likely to represent soluble vacuolar proteins. They have low molecular mass (around 20 kDa) and slightly acidic isoelectric points. In culture, evacuolated tobacco protoplasts regenerated a vacuole de novo. The vacuolation process as observed microscopically correlated welt with the reappearance of vacuolar marker enzymes. Likewise, the protein spots that were missing in the P(–)-pattern immediately after evacuolation reappeared within the first days of protoplast culture. By immunoblotting the same behaviour was demonstrated for the well-known vacuolar protein, tonoplast ATPase.     

Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle proteins in crassulacean Acid metabolism plants

Protoplasts and vacuoles were isolated and purified in large numbers from the CAM plants Ananas comosus (pineapple) and Sedum telephium for protein characterization. Vacuoles were further fractionated to yield a tonoplast vesicle preparation. Polypeptides of protoplasts, vacuoles, and tonoplast vesicles were compared to whole leaf polypeptides from both plants by one-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis. Approximately 100 vacuole polypeptides could be resolved of which 25 to 30% were enriched in the tonoplast vesicles. The proteins of protoplasts, vacuoles, and tonoplast vesicles from A. comosus were analyzed further by two-dimensional gel electrophoresis. When one-dimensional electrophoretograms of A. comosus polypeptides were stained with a glycoprotein-specific periodic acid Schiff stain, very few polypeptides appeared to be glycosylated, whereas a large number of glycosylated polypeptides were detected with a silver-based glycoprotein stain particularly in tonoplast vesicles. Analysis of the enzymic content of vacuoles from both plants indicated the presence of a variety of hydrolases, including bromelain as a major constituent of A. comosus. No substrate-specific ATPase, however, could be detected in vacuoles or tonoplast vesicles from either plant.     

Reappearance of hydrolytic activities and tonoplast proteins in the regenerated vacuole of evacuolated protoplasts

Mesophyll protoplasts of tobacco (Nicotiana tabacum L. cv. Xanthi) were evacuolated by centrifugation in a density gradient. Evacuolation resulted in the quantitative loss of vacuolar hydrolytic activities. The evacuolated miniprotoplasts were cultivated under different conditions, and the regeneration of the central vacuole was investigated by light and electron microscopy as well as by the determination of activities of vacuolar marker enzymes. Vacuoles and hydrolytic activities, as well as cell wall material reappeared faster when the cells were cultivated at low osmotic strength. A newly synthesized tonoplast polypeptide could be detected using a polyspecific serum raised against tonoplast proteins of barley (Hordeum vulgare L.). Both vacuolar proton pumps, the ATPase as well as the pyrophosphatase appear to be newly synthesized during the regeneration of the vacuole.Abbreviations GAP-DH NADP-dependent glyceraldehyde 3-phosphate dehydrogenase - PEP phosphoenolpyruvate - PPi pyrophosphate - PPase pyrophosphatase We thank Dr. Ernst Wehrli, Labor für Elektronenmikroskopie I, ETH Zürich, for taking micrographs. Esther Vogt assisted in the determination of the hydrolases. Bafilomycin was kindly provided by Professor Altendorf, Osnabrück FRG. This work was supported by the Swiss National Foundation grant No. 31-25196.88.     

Differential protein synthesis in response to sulphate and phosphate deprivation: Identification of possible components of plasma-membrane transport systems in cultured tomato roots

Isolated roots of Lycopersicon esculentum Mill., cultured in axenic conditions were starved of sulphate or phosphate, and uptake capacities for the respective oxyanion-transport systems were observed for several days after sulphate or phosphate withdrawal. Sulphate-uptake capacity of the intact roots, measured in a 20-min period, increased from a control level of 100 nmol · g^–1 · h^–1 to 1100 nmol · g^–1 · h^–1 in 10 d, and phosphate-uptake capacity increased from 500 to 1400 nmol · g^–1 · h^–1 over 4 d. Newly synthesised polypeptides of these root cultures were pulse-labelled in vivo for 2 h, by adding [³H]leucine to the culture medium. The tissue was immediately homogenised and soluble and membrane fractions were prepared. A highly purified plasma-membrane fraction was separated from the crude microsomal membrane fraction using an aqueous two-phase partitioning technique. All fractions were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. A 28-kilodalton (kDa) soluble polypeptide, and 36-, 43-, and 47-kDa plasma-membrane polypeptides were observed to have increased labelling after 4 d of sulphate deprivation. Longer periods resulted in additional polypeptides with increased [³H]leucine incorporation. The synthesis of a 25-kDa membrane polypeptide and a 65-kDa soluble polypeptide was increased after 4 d of phosphate deprivation. Two-dimensional electrophoresis afforded greater resolution of the plasmamembrane polypeptides, confirming increased synthesis of the 36-kDa polypeptide and the presence of the 28-kDa polypeptide in the plasma-membrane preparation from sulphate-starved roots. These polypeptides were also observed in protein-stained two-dimensional gels as low-abundant protein components of the plasmamembrane fraction. It is suggested that the 36-kDa polypeptide may be a component of the plasma-membrane sulphate-transport system and that the 25-kDa polypeptide may be a component of a phosphate-transport system.Abbreviations kDa kilodalton(s) - PAGE polyacrylamide gel electrophoresis - pI isoelectric point - SDS Sodium dodecyl sulphate This work was supported by the Agricultural and Food Research Council via grants-in-aid to Long Ashton Research Station. We are also grateful for discussions with our colleagues D.T. Clarkson (LARS) and J.-C. Davidian (ENSA/INRA, Montpellier).     

Characterization of the epidermis from barley primary leaves

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 m, whereas that of mesophyll protoplasts was 35.7 m. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [³⁵S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor U. Heber (Lehrstuhl Botanik 1, Würzburg) for his continuous support. This work was supported by the DFG and the University of Würzburg within the Sonderforschungsbereich 176.     

Reconstitution of the tonoplast amino-acid carrier into liposomes

The tonoplast amino-acid transporter of barley (Hordeum vulgare L.) mesophyll cells was functionally reconstituted by incorporating solubilized tonoplast membranes, vacuoplast membranes or tonoplast-enriched microsomal vesicles into phosphatidylcholine liposomes. (i) Time-, concentration- and ATP-dependence of amino-acid uptake were similar to results with isolated vacuoles. Although the orientation of incorporation could not be controlled, the results indicate that the transporter functions as a uniport system which allows regulated equilibration by diffusion between the cytosolic and vacuolar amino-acid pools. (ii) The ATP-modulated amino-acid carrier was also successfully reconstituted from barley epidermal protoplasts and Valerianella or Tulipa vacuoplasts, indicating its general occurrence. (iii) Fractionation of solubilized tonoplasts by size-exclusion chromatography followed by reconstitution of the fractions for glutamine transport gave two activity peaks: the first eluted in the region of high-molecular-mass vesicles and the second at a size of 300 kDa for the Triton-protein micelle.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacryl-amide gel electrophoresis This work was part of our research efforts within the Sonderforschungsbereich 176 of the University. We gratefully acknowledge experimental support by Marion Betz and valuable discussions with Professors U. Heber and U.-I. Flügge and Dr. Armin Gross (University of Würzburg) and Dr. E. Martinoia (ETH, Zürich, Switzerland).     

Identification of 3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate- and N,N'-dicyclohexylcarbodiimide-binding subunits of a higher plant H+-translocating tonoplast ATPase

The polypeptide composition of the NO-3-sensitive H+-ATPase of vacuolar membrane (tonoplast) vesicles isolated from red beet (Beta vulgaris L.) storage root was investigated by affinity labeling with [alpha-32P]3-O-(4-benzoyl)benzoyladenosine 5'-triphosphate [( alpha-32P]BzATP) and [14C]N,N'-dicyclohexylcarbodiimide [( 14C]DCCD). The photoactive affinity analog of ATP, BzATP, is a potent inhibitor of the tonoplast ATPase (apparent KI = 11 microM) and the photolysis of [alpha-32P]BzATP in the presence of native tonoplast yields one major 32P-labeled polypeptide of 57 kDa. Photoincorporation into the 57-kDa polypeptide shows saturation with respect to [alpha-32P]BzATP concentration and is blocked by ATP. [14C]DCCD, a hydrophobic carboxyl reagent and potent irreversible inhibitor of the tonoplast ATPase (k50 = 20 microM) labels a 16-kDa polypeptide in native tonoplast. The tonoplast ATPase is purified approximately 12-fold by Triton X-100 solubilization and Sepharose 4B chromatography. Partial purification results in the enrichment of two prominent polypeptides of 67 and 57 kDa. Solubilization, chromatography, and sodium dodecylsulfate-polyacrylamide gel electrophoresis of tonoplast labeled with [alpha-32P]BzATP or [14C]DCCD results in co-purification of the 57- and 16-kDa labeled polypeptides with ATPase activity. It is concluded that the tonoplast H+-ATPase is a multimer containing structurally distinct BzATP- and DCCD-binding subunits of 57 and 16 kDa, respectively. The data also suggest the association of a 67-kDA polypeptide with the ATPase.     

Cytosolic and cell-wall-bound acid invertases from leaves of Urtica dioica L.: a comparison

Two different forms of acid invertase (EC 3.2.1.26) were extracted from expanding leaves of the stinging nettle (Urtica dioica L.). One form was soluble and could be localized within the cytosol, whereas the other was ionically bound to the cell-wall and could not be detected in protoplasts. Both forms were purified, the latter to homogeneity. Western blotting with antibodies against the pure enzyme from cell walls was positive with the cell-wall enzyme but negative with the soluble form of acid invertase. Both forms are glycoproteins with identical molecular weights of 58 kDa. The K_m values for sucrose (raffinose) are 5 mM (4.8 mM) for the soluble and 1.2 mM (3.6 mM) for the cell-wall-bound enzyme. The pH optimum of the latter is slightly more acidic (4.5) than that of the soluble invertase (5.5). Both forms could easily be distinguished by their isoelectric points which were determined at pH 4.6 for the soluble and pH 9.3 for the wall-bound enzyme. When extraction and purification were carried out in the absence of protease inhibitors, both acid invertases showed microheterogeneity (multiple forms). However, with benzamidine and phenylmethylsulfonylfluoride as protease inhibitors each invertase produced only one protein band upon isoelectric focusing and gel electrophoresis, respectively.Abbreviations B benzamidine - Con A concanavalin A - FPLC fast protein liquid chromatography - IEF isoelectric focusing - kDa kilodalton - pI isoelectric point - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium dodecyl sulfate This work was supported by the Deutsche Forschungsgemeinschaft within the scope of the Sonderforschungsbereich 137.     

Simple method to isolate vacuoles and protoplasts for patch-clamp experiments

It was not possible to obtain protoplasts or vacuoles from the thallus of the liverwortConocephalum conicum by applying cell-wall-degrading enzymes. Therefore, a surgical method was developed to isolate protoplasts and vacuoles. A thallus was plasmolyzed and cut. The few protoplasts along the cutting edge that were not destroyed emerged from the edge under deplasmolysis and became thus accessible for a patch pipette. Whereas under slightly hypoosmolar conditions the emerging protoplast remained largely intact, more hypoosmolar conditions gave rise to isolated vacuoles. This method to isolate protoplasts and vacuoles could also be applied to other plant tissues like leaves ofArabidopsis thaliana. Patch-clamp measurements were performed with isolated vacuoles and excised tonoplast patches. A slowly activating vacuolar channel inC. conicum displayed the characteristic features of higher-plant slowly activating vacuolar channels.Abbreviations AP action potential - SV channel slowly activating vacuolar channel     

The effects of salt stress on polypeptides in membrane fractions from barley roots

Cell fractions enriched in endoplasmic reticulum, tonoplast, plasma membrane, and cell walls were isolated from roots of barley (Hordeum vulgare L. cv CM 72) and the effect of NaCl on polypeptide levels was examined by two-dimensional (2D) polyacrylamide gel electrophoresis. The distribution of membranes on continuous sucrose gradients was not significantly affected by growing seedlings in the presence of NaCl; step gradients were used to isolate comparable membrane fractions from roots of control and salt-grown plants. The membrane and cell wall fractions each had distinctive polypeptide patterns on 2D gels. Silver-stained gels showed that salt stress caused increases or decreases in a number of polypeptides, but no unique polypeptides were induced by salt. The most striking change was an increase in protease resistant polypeptides with isoelectric points of 6.3 and 6.5 and molecular mass of 26 and 27 kilodaltons in the endoplasmic reticulum and tonoplast fractions. Fluorographs of 2D gels of the tonoplast, plasma membrane, and cell wall fractions isolated from roots of intact plants labeled with [³⁵S]methionine in vivo also showed that salt induced changes in the synthesis of a number of polypeptides. There was no obvious candidate for an integral membrane polypeptide that might correspond to a salt-induced sodium-proton anti-porter in the tonoplast membrane.     

Characterization of the subunit structure of the maize tonoplast ATPase. Immunological and inhibitor binding studies

Gradient purified preparations of the maize 400-kDa tonoplast ATPase are enriched in two major polypeptides, 72 and 62 kDa. Polyclonal antibodies were prepared against these two putative subunits after elution from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel slices and against the solubilized native enzyme. Antibodies to both the 72- and 62-kDa polypeptides cross-reacted with similar bands on immunoblots of a tonoplast-enriched fraction from barley, while only the 72-kDa antibodies cross-reacted with tonoplast and tonoplast ATPase preparations from Neurospora. Antibodies to the 72-kDa polypeptide and the native enzyme both strongly inhibited enzyme activity, but the 62-kDa antibody was without effect. The identity and function of the subunits was further probed using radiolabeled covalent inhibitors of the tonoplast ATPase, 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole ([14C]NBD-Cl) and N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD). [14C]NBD-Cl preferentially labeled the 72-kDa polypeptide, and labeling was prevented by ATP. [14C]DCCD, an inhibitor of the proton channel portion of the mitochondrial ATPase, bound to a 16-kDa polypeptide. Venturicidin blocked binding to the mitochondrial 8-kDa polypeptide but did not affect binding to the tonoplast 16-kDa polypeptide. Taken together, the results implicate the 72-kDa polypeptide as the catalytic subunit of the tonoplast ATPase. The DCCD-binding 16-kDa polypeptide may comprise the proton channel. The presence of nucleotide-binding sites on the 62-kDa polypeptide suggests that it may function as a regulatory subunit.     

Identification with a photoaffinity reagent of a tonoplast protein involved in vacuolar malate transport of Catharanthus roseus

The effect of N-(4-azido-salicylyl) aspartic acid (AzSA), a photolysable analogue of malate, was tested on the malate transport activity of tonoplast vesicles isolated from Catharanthus roseus cell suspension cultures. AzSA inhibited malate uptake in a competitive manner with a K_ti of 1.7 millimolar. When iodinated, the malate analogue was found to be still photolysable and a competitive inhibitor of malate uptake. Photolysis of ¹²⁵I-labelled AzSA in the presence of purified tonoplast vesicles led to label incorporation into several polypeptides after analysis by gel electrophoresis. Only one polypeptide, with an apparent molecular mass of 37 kDa, was totally protected by the inclusion of 50 millimolar malate, the original substrate, in the photolysis medium. The labelled polypeptide is therefore apparently a specific malate-binding protein. Diethylpyrocarbonate (DEPC), a very potent inhibitor of malate transport acting at the active site of the transporter, also protected the 37 kDa polypeptide from labelling. Citrate and, to a lesser extent, quinate afforded protection from labelling whilst other organic acids or aspartic acid (100 millimolar) did not. These photoprotection results are in good agreement with the data concerning the specificity of malate transport across the tonoplast. Polyclonal antibodies against the 37 kDa polypeptide strongly inhibited malate uptake both in tonoplast vesicles and in isolated vacuoles. These results suggest the involvement of the 37 kDa polypeptide in vacuolar malate transport.     

Floral-specific polypeptides of the Japanese morning glory

Polypeptides from stems, leaves, sepals, corollas, stamens and pistils of the Japanese morning glory (Ipomoea nil Roth (Pharbitis nil Chois.)) were separated by one- and two-dimensional gel electrophoresis and visualized by silver staining. The majority of polypeptides were expressed in two or more organs, while those specific to only one organ were comparatively rate. Among the polypeptides of the former class were two which appeared to be floral-specific. A 46-kDa (kilodalton) polypeptide was expressed in corollas, stamens and pistils, whereas a 32-kDa polypeptide was observed only in extracts prepared from reproductive organs. Polypeptide spots from the various organs were compared with those from leaves, and it was found that sepals and stems shared 40–50% of their polypeptides with leaves, whereas corollas, stamens and pistils shared 20% or less. The latter organs shared 120 polypeptides or roughly 15% of those identified in the floral extracts. Floralorgan-specific polypeptides comprised nearly 10% of the total floral polypeptides identified.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate     

Unassembled polypeptides of the plastidic ribosomes in heat-treated 70S-ribosome-deficient rye leaves

The polypeptides of the subunits of 70S ribosomes isolated from rye (Secale cereale L.) leaf chloroplasts were analyzed by two-dimensional polyacrylamide gel electrophoresis. The 50S subunit contained approx. 33 polypeptides in the range of relative molecular mass (M_r) 13000–36000, the 30S subunit contained approx. 25 polypeptides in the range of M_r 13000–40500. Antisera raised against the individual isolated ribosomal subunits detected approx. 17 polypeptides of the 50S and 10 polypeptides of the 30S subunit in the immunoblotting assay. By immunoblotting with these antisera the major antigenic ribosomal polypeptides (r-proteins) of the chloroplasts were clearly and specifically visualized also in separations of leaf extracts or soluble chloroplast supernatants. In extracts from rye leaves grown at 32° C, a temperature which is non-permissive for 70S-ribosome formation, or in supernatants from ribosome-deficient isolated plastids, six plastidic r-proteins were visualized by immunoblotting with the anti-50S-serum and two to four plastidic r-proteins were detected by immunoblotting with the anti-30S-serum, while other r-proteins that reacted with our antisera were missing. Those plastidic r-proteins that were present in 70S-ribosome-deficient leaves must represent individual unassembled ribosomal polypeptides that were synthesized on cytoplasmic 80S ribosomes. For the biogenesis of chloroplast ribosomes the mechanism of coordinate regulation appear to be less strict than those known for the biogenesis of bacterial ribosomes, thus allowing a marked accumulation of several unassembled ribosomal polypeptides of cytoplasmic origin.Abbreviations L polypeptide of large ribosomal subunit - M_r relative molecular mass - r-protein ribosomal polypeptide - S polypeptide of small ribosomal subunit - SDS sodium dodecyl sulfate     

Basic peroxidases in isolated vacuoles of nicotiana tabacum L.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).Abbreviation ER endoplasmic reticulum - PAGE polyacrylamide gel electrophoresis     

An examination of the peroxidases from Lupinus albus L. hypocotyls

Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one- and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligand-induced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60 of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pIs 9.5, 9.7 and >9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implications to current understanding of peroxidases in L. albus are discussed.Abbreviations APAGE anionic polyacrylamide gel electrophoresis - CA coniferyl alcohol - CPAGE cationic polyacrylamide gel electrophoresis - IEF isoelectric focusin - NEIEF non-equilibrated isoelectric focusing - 2D two dimensional - pI isoelectric point - RCPAGE reversed current polyacrylamide gel electrophoresis     

Particle density and protein composition of the peribacteroid membrane from soybean root nodules is affected by mutation in the microsymbiont Bradyrhizobium japonicum

Particle frequency of the peribacteroid membrane (PBM) from nodules of Glycine max (L.) Merr. cv. Maple Arrow infected with Bradyrhizobium japonicum 61-A-101 (wild-type strain) was determined by freeze-fracturing to be about 2200·m^-2 in the protoplasmic fracture face and 700·m^-2 in the exoplasmic fracture face. In membranes isolated from nodules infected with the mutant RH 31-Marburg of B. japonicum, the particle frequency was similar in both fracture faces with 1200–1300 particles·m^-2. Analysis of particlesize distribution on peribacteroid membranes showed a loss, especially of particle sizes larger than 11 nm, in the mutant-infected nodules. Two-dimensional gel electrophoresis (isoelectric focussing and sodium dodecyl sulfate-polyacrylamide) showed 27 different polypeptides in the PBM from nodules infected with the wild-type strain, four of which were absent from the PBM of nodules infected with the mutant RH 31-Marburg, which also exhibited one extra small-molecular-weight polypeptide. At least 14 of the 27 polypeptides in the PBM from the wild-type-infected nodule were glycoproteins. In three of these glycoproteins, post-translational modifications were either lacking or different when the membrane was derived from mutant-infected nodules.Abbreviations EF exoplasmatic fracture face - HRPO horse radish peroxidase - IEF Isoelectric focussing - PBM peribacteroid membrane - PF protoplasmatic fracture face - PNA peanut agglutinin - PSA Pisum sativum agglutinin - SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

An analysis of vacuole development in oat aleurone protoplasts

Cultured oat (Avena sativa L. — naked form) aleurone protoplasts were employed as a model system for following changes which accompany the development of vacuoles during in-vitro incubation. Over a 5-d period, the aleurone grains progressively grew and fused to form a large central vacuole and the volume of the protoplasts increased sevenfold. The growth of the vacuole was accompanied by a progressive acidification of the vacuolar sap. Vacuolation was inhibited by high concentrations of mannitol and by cycloheximide and cordycepin applied at various times during the incubation period. Neither cycloheximide nor cordycepin affected the initial phases of vacuolation but cycloheximide retarded subsequent stages, particularly if added early in the incubation. Cordycepin inhibited only the later stages of vacuolation. Radiolabelling studies identified at least three novel microsomal proteins, with relative molecular masses of approximately 34, 47 and 48 kDa, which appeared during vacuolation and whose synthesis was markedly affected by these inhibitors.Abbreviations CF carboxyfluorescein - CFDA 6 carboxyfluorescein diacetate - TIP tonoplast intrinsic protein We are grateful to Dr Richard Hooley and Dr Robert Walker (Long Ashton Research Station) for providing the methodology for aleurone protoplast isolation and to Professeur Francis Marty (Université de Bourgogne, Dijon) for providing antibodies to the red beet TIP. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.     

A phloem-specific,lectin-like protein is located in pine sieve-element plastids by immunocytochemistry

Two co-purifying phloem polypeptides of 24 and 25 kilodaltons (kDa) were isolated from homogenates of Pinus sabiniana Dougl. phloem by differential centrifugation, selective solubilization and electrophoresis, and rabbit antibodies raised against them. The antisera were found to be specific for doublet bands between 23 and 25 kDa in Western blots of whole phloem extracts of Pinus species; no xylem polypeptides were labelled, nor did labelling occur in blots of phloem extracts from other genera in the Pinaceae. Solubilized phloem polypeptides bind strongly to chitin (oligomeric N-acetylglucosamine) columns and are sensitive to thiol reagents, both characteristics which relate them to phloemspecific lectins isolated from angiosperm species (C. Allen, 1979, Biochem. J. 183, 133–137; A.K. Gietl et al., 1979, Planta 144, 367–371). Fluorescence microscopy and immuno-gold electron microscopic cytochemistry demonstrated antigenic sites specifically associated with protein crystals peculiar to the sieve-element plastids of the Pinaceae.Abbreviations DAB diamino benzidine tetrachloride - FITC fluorescein isothiocyanate - kDa kilodalton - PBS phosphate-buffered saline - PP phloem polypeptide(s) - SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol