Nicotine-induced inhibition of lycopene cyclization in Phaseolus vulgaris cotyledons

The complete nicotine inhibition of lycopene cyclization during light-induced carotenogenesis in excised bean cotyledons was achieved. The inhibitory effect was easily reversible and removal of nicotine has allowed synthesis of the normal cyclic carotenoids.     

Proteolytic activities in Phaseolus vulgaris cotyledons under copper stress

The changes in the protease activities of bean cotyledons were investigated in response to copper stress. Assays using synthetic substrates and specific protease inhibitors followed by activity measurements and electrophoresis analysis allowed to study the classes of enzymes involved in the storage protein mobilization during the germination of bean (Phaseolus vulgaris L) seeds, and then identify which ones were affected in the presence of 200 μM CuCl₂ in the imbibition medium. Copper treatment affected embryo growth and total protease activity. The results of SDS-gelatin-PAGE show that Cu excess led to a decrease in protease activity of 45 to 66 kDa. Moreover, cysteine-, aspartic- and metallo-protease activities were markedly lowered under copper stress, while serine-protease one was enhanced as well as its activity dependent abundance in comparison with control. However, the relative distribution of major cysteine protease in H₂O-germinated seeds was significantly diminished after Cu exposure. Thus, copper excess can disturb the nitrogen freeing from reserve tissues at enzymatic level; differential responses of protease classes are discussed, notably, cysteine protease in the way of storage protein mobilization and serine protease in protective mechanism one.     

Synthesis of an integral protein of the protein-body membrane in Phaseolus vulgaris cotyledons

Protein-body membranes (PBMs) were isolated from cotyledons of Phaseolus vulgaris L. by a procedure involving osmotic shock of purified protein bodies. The purified PBMs have a characteristic density of 1.16 g cm^-3. Treatment of the membranes with increasing concentrations of detergent (Triton X-100) or with a solution at pH 12.0 showed that the membranes contained a characteristic integral protein (IMP) with a relative molecular mass of 25,000. This IMP is not a glycoprotein. When developing cotyledons were labeled with ³H-amino acids for 2–3 h, a radioactive polypeptide with the same mobility on denaturing polyacrylamide gels as IMP was found to be associated with the rough endoplasmic reticulum (ER). During a 24-h chase, a considerable portion of the radioactivity slowly transferred into the IMP associated with more rapidly sedimenting organelles, which sedimented in the same region of the sucrose gradients as the PBMs. Antibodies prepared against purified IMP crossreacted with an ER-associated protein which had the same mobility on denaturing acrylamide gels as authentic IMP. Synthesis of IMP occurred at all stages of cotyledon development examined, but not during seed germination. The results show that a newly synthesized protein of the PBM is associated with the rough ER, just like the soluble matrix proteins, phaseolin (R. Bollini, W. Van der Wilden and M.J. Chrispeels, 1983, J. Cell Biol. 96,999–1007) and phytohemagglutinin (M.J. Chrispeels and R. Bollini, 1982, Plant Physiol. 70, 1425–1428), but that the chase-out from the ER is much slower for IMP than for the matrix proteins.Abbreviations EDTA ethylenediamino-tetraacetic acid - ER endoplasmic reticulum - IMP integral membrane protein - PB protein body - PBM protein-body membrane - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Expression of glutamine-synthetase genes in cotyledons of germinating Phaseolus vulgaris L.

In the legume Phaseolus vulgaris L., glutamine synthetase (GS; EC.6.3.1.2.) is encoded by four actively transcribed genes, gln-, gln-, gln- and gln-. We have studied the expression of these genes in cotyledons during seed germination and have studied the effect of light and nitrate on this process. An RNase-protection method, used to detect the abundances of GS mRNAs, revealed that the four GS genes are differentially expressed in the germinating cotyledons. The gln-. mRNA was present in dry seeds and was the most abundant GS mRNA during early stages of germination. The gln- and gln- mRNAs were first detectable 2 d after sowing and their abundances differed in light- and dark-grown cotyledons at later stages of germination. The gln- mRNA (which encodes the plastid-located GS) was detectable only in light-grown cotyledons, at a low abundance. A nitrate supply of 2 mM had only a minor effect on the expression of the GS genes. Western immunodetection and ion-exchange high-performance liquid chromatography demonstrated that the polypeptide and isoenzyme were present in extracts of dry seeds and represented the major GS products at 2 d and 4 d. Both the and polypeptides appeared at the 2-d stage. The role of differential GS gene expression in controlling cotyledonary GS activity is discussed.Abbreviations 1D, 2D one-, two-dimensional - GS glutamine synthetase - GS_t GS transferase activity - IEX-HPLC ion-exchange high-performance liquid chromatography - kDa kilodaltons - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We are grateful to the Association of Commonwealth Universities and the Science and Engineering Research Council for financially supporting R.S. and to the S.E.R.C. for a grant to support M.J.B. We would like to thank Dr K.J.F. Farnden (University of Otago, New Zealand) and Dr T.H.N. Ellis (John Innes Institute, Norwich) for scanning the autoradiographs for Fig. 2.     

Regulation of chlorophyll and Rubisco levels in embryonic cotyledons of Phaseolus vulgaris

While deep within the maternal tissues (pods and testa), cotyledons of the bean (Phaseolus vulgaris L.) green and the plastids differentiate as chloroplasts. At the time of seed maturation the chloroplasts dedifferentiate and the green color is lost. We have used Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and chlorophyll to study chloroembryo development. Chlorophyll levels and Rubisco activity increase early in embryonic development then decline as the cotyledons enter the maturation phase. Rubisco accumulation follows a strong temporal pattern over the course of embryo development, and furthermore, occurs in total darkness. Therefore, accumulation of Rubisco during embryogenesis may occur in response to developmental signals. In embryos developed in total darkness, Rubisco accumulation was uncoupled from chlorophyll accumulation. Exposure of isolated cotyledons to abscisic acid (ABA) resulted in loss of chlorophyll and decline in Rubisco levels comparable to those seen in normal embryogenesis. This indicates that the decline in Rubisco in chloroembryos in vivo results from factors such as ABA that signal the onset of maturation. The results show that ABA not only enhances the accumulation of some proteins (e.g. storage proteins), but also depresses the accumulation of others during embryogeny.Abbreviations Rubisco ribulose-1,5-bisphosphate-carboxylase/oxygenase (EC 4.1.1.39) - LSU large subunit of Rubisco - SSU small subunit of Rubisco - ABA abscisic acid - FW fresh weight     

The compartmentation of ethylene in developing cotyledons of Phaseolus vulgaris L.

Isolated cotyledons of Phaseolus vulgaris L. cv. Canadian Wonder accumulated ¹⁴C₂H₄ (0.7–1 l l^-1) from air to give partition coefficients of 1 to 4, which greatly exceeded the value obtained with steam killed cotyledons (0.05) and with water (0.11). After ¹⁴C₂H₄ treatment, 98% of the ¹⁴C in the tissue remained as ¹⁴C₂H₄. The labelled ethylene accumulated by cotyledons was released only slowly (1–10% h^-1) either in an air stream or into toluene. Heating to 60°C for 2 h, but not freezing and thawing, caused the immediate release of ¹⁴C₂H₄ from the tissue. Propylene and vinyl chloride competitively inhibited the accumulation of ¹⁴C₂H₄.Cotyledons emanated endogenous ethylene at a very low rate but after heating (although not freezing and thawing) 13 nl of ethylene per g fresh mass were released within minutes. It was concluded that french bean cotyledons hold ethylene in a compartmented form in sufficient amount to account for at least 200 h of emanation.Abbreviation PPO diphenyloxazole     

Partial purification of an ethylene-binding site from Phaseolus vulgaris L. cotyledons

The solubilised ethylene-binding site (EBS) of Phaseolus vulgaris L. cotyledons is an asymmetrical protein with a sedimentation coefficient of 2 S and a Stoke's radius of 6.1 nm (determined by ultracentrifugation on isokinetic gradients and gel-permeation chromatography, respectively). The molecular weight and frictional ratio were calculated as 52 000–60 000 and 2.37–2.48, respectively. The EBS has an isoelectric point at between pH 3–5, determined by isoelectric focussing and exhibits a negative charge at pH 8 during non-denaturing electrophoresis. The electrical charge on the EBS is shielded; the EBS does not bind to anion-exchange media under the experimental conditions reported here, is not precipitated by ammonium sulphate and does not precipitate at its isoelectric pH. The EBS preferentially partitions into detergent phases. The results indicate that the EBS is a hydrophobic protein complexed with detergent in aqueous solution. The techniques used to characterise the EBS also resulted in varying degress of purification.Abbreviations EBS ethylene-binding site - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Conidiogenic effects of mannose-binding lectins isolated from cotyledons of red kidney bean (Phaseolus vulgaris) on Alternaria alternata

Effect of proteinaceous extracts from red kidney bean cotyledons on mycelium of Alternaria alternata growing on potato dextrose agar (PDA) plates was investigated. Unexpectedly, conidia formation was induced in response to applied crude extracts. A PDA disc method was developed to quantify conidia formed. A purified fraction retaining conidiation inducing effect (CIE) was obtained following several protein purification procedures including the last step of eluting bound proteins from an Affi-gel blue gel column. Based on MALDI (matrix assisted laser desorption/ionization) mass spectrometric analysis, a previously identified mannose-binding lectin (MBL) called PvFRIL (Phaseolus vulgaris fetal liver tyrosine kinase 3-receptor interacting lectin) was present in this conidiation inducing fraction. The PvFRIL was subsequently purified using a single step mannose-agarose affinity column chromatography. When the lectin was applied exogenously to A. alternata, increased conidiation resulted. The conidia produced in response to the MBL were similar to those induced by other methods and their germ tubes were longer after 12 h growth than those induced under white light. To our knowledge this is the first report of exogenous application of a PvFRIL or another purified protein from a plant inducing conidia formation in a fungus.     

Incorporation of fucose into the carbohydrate moiety of phytohemagglutinin in developing Phaseolus vulgaris cotyledons

Incubation of developing cotyledons of P. vulgaris with [³H]fucose resulted in the incorporation of radioactivity into the cell wall, membranous organelles and soluble macromolecules. Fractionation of the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by fluorography, showed that phytohemagglutinin (PHA) was the major fucosylated protein synthesized in the cotyledons. Incorporation of fucose into PHA occurred in the membranous organelle fraction, and the radioactive fucose remained associated with the PHA during a 20-h chase of the radioactivity. Tunicamycin inhibited the incorporation of glucosamine and fucose into PHA to the same extent (65%), indicating the involvement of a lipid intermediate in the incorporation of fucose, or the attachment of fucose to the high-mannose oligosaccharide moiety of newly synthesized PHA. Digestion with proteinase K of [³H]fucose- or [³H]glucosamine-labeled PHA resulted in the formation of glycopeptides of similar size. These glycopeptides were partially resistant to digestion with endo-β-N-acetylglucosaminidase H, even after the removal of fucose by mild acid hydrolysis. We postulate, on the basis of these experiments, that the transport of PHA from the endoplasmic reticulum to the protein bodies is accompanied by the modification of its oligosaccharide side-chain. This modification involves inter alia the attachment of fucose, and renders the oligosaccharide side-chain resistant to digestion with endo-β-N-acetylglucosaminidase H. Analogy with animal glycoproteins indicates that this modification probably occurs in the Golgi apparatus.     

A promotive action of kinetin on amylase activity in cotyledons of Phaseolus vulgaris

Kinetin and the embryo axis acted similarly in bringing abouta promotion of amylase activity in cotyledons of Phaseolus vulgaris.No promotive effect of gibberellic acid or indole-3-acetic acidon amylase activity could be detected. It is suggested thatthe regulatory action of the embryo axis on starch degradationin the cotyledons of P. vulgaris is mediated by cytokinins. (Received May 4, 1970; )     

Mevalonate kinase in green leaves and etiolated cotyledons of the French bean Phaseolus vulgaris

1. Partially purified preparations of mevalonate kinase were obtained from green leaves and etiolated cotyledons of Phaseolus vulgaris. 2. After removal of interfering polyphenols both enzyme preparations behaved identically on gel filtration, ion-exchange chromatography and density-gradient centrifugation. 3. The kinetic parameters of the preparations from the two sources were indistinguishable. The preparation from etiolated cotyledons had a K(m) of 4.26x10(-5)m for RS-mevalonate and 1.54x10(-3)m for ATP. The preparation from green leaves had a K(m) of 4.55x10(-5)m for RS-mevalonate and 1.75x10(-3)m for ATP. The pH optimum of both enzyme preparations was pH7.0. 4. The effect of inhibitors on the two enzyme preparations was similar, both being inhibited by reagents known to react with thiol groups, and the two preparations had similar inhibitor constants for competitive inhibition by prenyl pyrophosphates. 5. The molecular weight of the enzyme in both preparations was estimated to be 100000; the enzymes from the two preparations had similar mobilities on polyacrylamide-gel electrophoresis.     

The rough endoplasmic reticulum is the site of reserve-protein synthesis in developing Phaseolus vulgaris cotyledons

Cytyledons of the common bean, Phaseolus vulgaris L., were incubated with radioactive amino acids at different stages of seed development. The proteins were fractionated by ion-exchange chromatography, sucrose gradients, and sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis. From 16 to 28 d after flowering about 40% of the incorporated radioactivity was associated with the polypeptides of vicilin and 10% with those of phytohemagglutinin.Polysomes were isolated from developing cotyledons 20–25 d after flowering and free polysomes were separated from membrane-bound polysomes. Aurintricarboxylic acid, an inhibitor of initiation in cell-free translation systems, did not inhibit the incorporation of amino acids into in-vitro synthesized proteins, indicating that synthesis was limited to the completion of already initiated polypeptides. Autofluorography of SDS-polyacrylamide gels showed that the two classes of polysomes made two different sets of polypeptides and that there was little overlap between these two sets.Four polypeptides similar in size to the 4 polypeptides of vicilin were made by membrane-bound polysomes and not by free polysomes. Antibodies specific for vicilin bound to those 4 polypeptides. Free polysomes made only polypeptides which did not bind to antibodies specific for vicilin. Antibodies against phytohemagglutinin did not bind to any of the invitro synthesized polypeptides.The membranes to which the polysomes were bound were characterized on sucrose gradients and by electron microscopy. Polysomes recovered from membranes which banded on top of 35 and 50% sucrose synthesized the vicilin polypeptides most rapidly. These membrane fractions were rich in vesicles of rough endoplasmic reticulum (ER). The ER marker-enzyme NADH-cytochrome-c reductase banded with an average density of 1.18 g/cm³ (40% w/w sucrose) on continuous gradients. These experiments demonstrate that the ER is the site of vicilin synthesis in developing bean cotyledons. Quantitative determinations of several ER parameters (RNA and lipid-phosphate content, NADH-cytochrome-c-reductase activity) show that expansion of the cotyledons is accompanied by a 4-6-fold increase in ER.     

Properties of satellite DNA of Phaseolus vulgaris]

Nuclear DNA components of Ph. vulgaris were preparatively separated by equilibrium ultracentrifugation in Hg++-Cs2SO4 density gradient (buoyant density of the major component in CsCl density gradient 1.694 g/cm3., satellite component--1.703 g/cm3). The properties of individual DNA fractions were investigated. The melting curve of satellite DNA of Ph. vulgaris has biphasic character. The observed heterogeneity of satellite DNA component is of intermolecular nature. This is illustrated by the splitting of unsheared satellite DNA into two components during renaturation, as well as by its behaviour in Hg++-Cs2SO4 density gradient at high rf value. The width of satellite DNA reassociation curve covers three decades of Cot. The length of the major repeating sequences of the satellite component is close to the length of phage T2 DNA. During chromatography on MAK column satellite DNA elutes earlier than the major component due to its higher GC-content. It is suggested that one of the satellite DNA fractions of Ph. vulgaris contains rRNA genes.     

Purification of an endopeptidase involved with storage-protein degradation in Phaseolus vulgaris L. cotyledons

Phaseolin, the major seed storage protein of Phaseolus vulgaris L., is degraded in the cotyledons in the first 7–10 d following seed germination. We assayed cotyledon extracts for protease activity by using [³H]phaseolin as a substrate and then fractionated the digestion mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in order to identify the cleavage products. The cotyledons of 4-d-old seedlings contain an endopeptidase which cleaves the polypeptides of [³H]phaseolin (apparent molecular weights=51 000, 48 000, 46 000 and 43 000) into three discrete clusters of proteolytic fragments (M _rs=27 000, 25 000 and 23 000). Endopeptidase activity is not detected in the cotyledons until the protein content of these organs starts to decline, shortly after the first day of seedling growth. Endopeptidase activity increases to a maximum level in the cotyledons of 5-d-old seedlings and then declines to a minimum value by day 10. The enzyme was purified 335-fold by ammonium-sulfate precipitation, organomercurial-agarose chromatography, gel filtration and ion-exchange chromatography. The endopeptidase constitutes 0.3% of the protein content in the cotyledons of 4-d-old seedlings. It is a cysteine protease with a single polypeptide chain (M _r=30 000). Optimum hydrolysis of [³H]phaseolin occurs at pH 5. The enzyme is irreversibly inactivated at pH values above 7 and at temperatures above 45° C. The endopeptidase attacks only a limited number of peptide bonds in [³H]phaseolin, without causing any appreciable change in the native molecular weight of the storage protein. The endopeptidase is also able to hydrolyze the bean-seed lectin, phytohemagglutinin. Thus, this enzyme may play a general role in degrading cotyledon proteins of P. vulgaris following seed germination.Abbreviations Da dalton - DTT dithiothreitol - M _r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - PHA phytohemagglutinin - SDS sodium dodecyl sulfate     

Seed reserve-protein glycosylation in an in vitro preparation from developing cotyledons of Phaseolus vulgaris

A particulate preparation from developing cotyledons of Phaseolus vulgaris L. was incubated with uridine-5-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc; [6-³H]glucosamine), and by polyacrylamide gel electrophoretic analysis it was shown that the labeled (N-acetyl)glucosamine (GlcNAc) was incorporated into the principal reserve protein of the cotyledons, vicilin, and also into phytohemagglutinin. Some of the labeled product also reacted with antiserum to vicilin from mature seeds. In contrast it was not possible to detect the incorporation of labeled mannose from guanosine-5-diphospho-D-mannose (GDP-mannose; [U-¹⁴C]mannose) into either of these proteins by gel-electrophoretic analysis of the mannose-labeled products, but we did observe a low incorporation of mannose into material which reacted with antiserum to vicillin. The predominant glycosylation reaction in vitro was therefore probably a transfer of GlcNAc alone, rather than in combination with mannose as preformed oligosaccharide.Abbreviations GlcNAc N-acetyl-D-glucosamine - GDP guanosine 5-diphospho - IEF isoelectric focusing - PHA phytohemagglutinin - SDS sodium dodecylsulfate - UDP uridine-5-diphospho