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.     

Immunocytochemical localization of phaseolin and phytohemagglutinin in the endoplasmic reticulum and Golgi complex of developing bean cotyledons

Development of legume seeds is accompanied by the synthesis of storage proteins and lectins, and the deposition of these proteins in protein-storage vacuoles (protein bodies). We examined the subcellular distribution, in developing seeds of the common bean, Phaseolus vulgaris L., of the major storage protein (phaseolin) and the major lectin (phytohemagglutinin, PHA). The proteins were localized using an indirect immunocytochemical method in which ultrathin frozen sections were immunolabeled with rabbit antibodies specific for either PHA or phaseolin. Bound antibodies were then localized using goat-anti-rabbit immunoglobulin G adsorbed onto 4- to 5-nm colloidal gold particles. The sections were post-fixed with OsO₄, dehydrated, and embedded in plastic on the grids. Both PHA and phaseolin exhibited a similar distribution in the storage-parenchyma cells, being found primarily in the developing protein bodies. Endoplasmic reticulum and Golgi complexes (cisternal stacks and associated vesicles) also were specifically labeled for both proteins, whereas the cytosol and other organelles, such as mitochondria, were not. We interpret these observations as supporting the hypothesis that the transport of storage proteins and lectins from their site of synthesis, the rough endoplasmic reticulum, to their site of deposition, the protein bodies, is mediated by the Golgi complex.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G - PBS phosphate-buffered saline - PHA phytohemagglutinin     

Characterization and subcellular localization of vicilin and phytohemagglutinin,the two major reserve proteins of Phaseolus vulgaris L.

Extracts of bean (Phaseolus vulgaris L. cv. Greensleeves) cotyledons contained two abundant proteins: vicilin and phytohemagglutinin. Vicilin, a 6.9 S protein fraction at neutral pH, associated to an 18.0 S form at pH 4.5 and had 3 non-identical subunits with molecular weights (MW) of 52,000, 49,000 and 46,000. Phytohemagglutinin, a 6.4 S protein fraction, had 2 non-identical subunits with MW of 34,000 and 36,000. Phytohemagglutinin could be separated by isoelectrofocusing into a mitogenic and non-erythroagglutinating protein with a single subunit of MW=34,000, and a mitogenic and erythroagglutinating protein fraction which contained both subunits. Vicilin is apparently identical with the so called glycoprotein II (A. Pusztai and W.B. Watt, Biochim. Biophys. Acta 365, 57–71, 1970) and with globulin G1 (R.C. McLeester, T.C. Hall, S.M. Sun, F.A. Bliss, Phytochem. 2, 85; 1973), while phytohemagglutinin is identical with globulin G2 (McLeester et al., 1973). Since vicilin and phytohemagglutinin are internationally used names there is no need to introduce new names to describe P. vulgaris reserve proteins. Both proteins are catabolized in the course of seedling growth and are located in the protein bodies, indicating that they are reserve proteins. Vicilin isolated in its 18.0 S form from the cotyledons of young seedlings contains substantial quantities of smaller polypeptides, in addition the 3 original ones. We suggest that the presence of these small polypeptides represents partial breakdown of the vicilin prior to its complete catabolism.     

The endoplasmic reticulum of mung-bean cotyledons

Germination and seedling growth of mungbean (Vigna radiata (L.) Wilczek) are accompanied by the incorporation of radioactive amino acids, glycerol, galactose, and glucosamine in an organelle fraction of the cotyledons which co-equilibrates with NADH-cytochrome-c-reductase activity at 1.13 g·cm^–3 on isopycnic gradients containing 1 mM EDTA. Up to 20% of the newly synthesized proteins accumulate in this organelle fraction. The organelle fraction has been identified as rough endoplasmic reticulum (ER) on the basis of its increased density (1.16 g·cm^–3) when 3 mM MgCl₂ is included in all media. Seedling growth is also accompanied by a marked rise (more than 5-fold) in ER-associated NADH- and NADPH-cytochrome-c-reductase activity, and by the incorporation of⁵⁹Fe into ER-associated heme. Other manifestations of the reorganization of the ER in the cotyledons include a relative increase in membrane-associated RNA (from 12% of total RNA after 12 h of imbibition to 23% after 6 d of growth), and a change in the pattern of polypeptides associated with the ER. These results provide further evidence for the extensive reorganization of the ER of the cotyledons which accompanies seedling growth. The reorganization includes the simultaneous breakdown of the pre-existing tubular ER and the biosynthesis of new ER components.This is the fourth paper in a series on the endoplasmic reticulum of mung-bean cotyledons. The first three papers are referenced as Gilkes and Chrispeels (in press); Harris and Chrispeels 1980; Van der Wilden et al. (in press)     

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     

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     

Crosslinking of microsomal proteins identifies P-9000, a protein that is co-transported with phaseolin and phytohemagglutinin in bean cotyledons

Developing cotyledons of the common bean, Phaseolus vulgaris L., transport within their secretory system (endoplasmic reticulum and Golgi apparatus) the abundant vacuolar proteins, phaseolin and phytohemagglutinin. To identify proteins that may play a role in vacuolar targeting, we treated cotyledon microsomal fractions with a bifunctional crosslinking reagent, dithiobis(succinimidyl propionate), isolated protein complexes with antibodies to phaseolin and phytohemagglutinin, and analysed the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis. This allowed us to identify a protein of M_r=9000 (P-9000) that was crosslinked to both phaseolin and phytohemagglutinin. P-900 is abundantly present in the endoplasmic reticulum. The aminoterminus of P-9000 shows extensive sequence identity with the amino-terminus of PA1 (M_r=11 000), a cysteine-rich albumin whose processing products accumulate in the vacuoles of pea (Pisum sativum L.) cotyledons. Like PA1, P-9000 is synthesized as a pre-proprotein that is posttranslationally processed into smaller polypeptides. The possible functions of P-9000 are discussed.Abbreviations DSP dithiobis(succinimidyl propionate) - EDTA ethylenediaminetetraacetic acid - ER endoplasmic reticulum - kDa kilodalton - M_r relative molecular mass - PHA phytohemagglutinin - SDS sodium dodecylsulfate - PAGE polyacrylamide gel electrophoresis     

Immunocytochemical localization of concanavalin A in developing jack-bean cotyledons

The lectin, concanavalin A (Con A), was localized in the cotyledon of developing jack beans (Canavalia ensiformis (L.) DC) by electron-microscope immunocytochemistry. In mature seeds, Con A was present in protein-storage vacuoles (protein bodies) of storage-parenchyma cells. Although protein bodies could be seen in other cell types, only protein bodies in storage-parenchyma cells contained Con A. During seed development, Con A was also localized on the endoplasmic reticulum and Golgi apparatus, presumably en route toward deposition within the protein bodies. The intensity of labeling of the endoplasmic reticulum was much greater during the developmental stage of protein-body filling (66% final seed weight) than in mature seeds.Abbreviations Con A concanavalin A - ER endoplasmic reticulum - IgG immunoglobulin G     

A study of the subcellular localisation of an ethylene binding site in developing cotyledons of Phaseolus vulgaris L. by high resolution autoradiography

Use was made of light microscopy and high resolution electron microscope autoradiography to determine the subcellular localisation of a binding site with a high affinity and specificity for ethylene in developing cotyledons of Phaseolus vulgaris L. The results indicate that the binding site is located on the endoplasmic reticulum and protein body membranes, confirming previous studies using cellular fractionation and marker enzymes.     

Immunocytochemical localization of allergenic protein (Ole e I) in the endoplasmic reticulum of the developing pollen grain of olive (Olea europaea L.)

The monoclonal antibody OL-1 and transmission electron microscopy were used to locate immunologically the major allergen of olive pollen. Ole e I, during pollen grain development. Within the pollen grain, allergenic proteins are located in the cisternae of the rough endoplasmic reticulum. Our findings indicate that the synthesis of these proteins starts in the vegetative cytoplasm of olive pollen during the early maturation stage.Dedicated to Professor Andreas Sievers on the occasion of his retirement     

The endoplasmic reticulum of mung-bean cotyledons: Quantitative morphology of cisternal and tubular ER during seedling growth

The ultrastructure of the endoplasmic reticulum (ER) in storage parenchyma cells in the cotyledons of mung beans (Vigna radiata L.) was examined during germination and seedling growth. Two different methods were used to visualize the ER: thin (0.08 m) sections of tissue fixed in formaldehyde and glutaraldehyde and post-fixed with osmium tetroxide, and thick (1 m) sections of tissue fixed in buffered aldehyde and post-fixed with zinc iodide-osmium tetroxide (ZIO). Changes in relative amounts of ER were quantified by morphometry (stereology).The ER occurs in two forms: a cisternal form with associated ribosomes which can be seen at all stages from imbibition to cotyledon senescence, and a tubular form which initially has associated ribosomes. Stereoscopic images of thick sections of cotyledons of 2-day-old seedlings show that the tubular ER consists of a three-dimensional array of interconnecting tubules which have numerous connections with the cisternal ER. The network of tubules and cisternae extends throughout the cytoplasm enveloping the protein bodies. Germination and seedling growth are accompanied by a reduction in the total volume occupied by the ER. This reduction is the result of a preferential loss of tubular ER and occurs largely before protein mobilization. Cisternal ER decreases during the first 48 h of imbibition and seedling growth, but storage cells subsequently show an increase in cisternal ER just prior to and during the period of protein mobilization. Cisternal ER remains conspicuous during the last phase of reserve mobilization when starch is broken down and the cells are starting autophagy.Abbreviations ER endoplasmic reticulum - ZIO zinc iodide-osmium tetroxide This is the second in a series of papers on the endoplasmic reticulum of mung bean cotyledons. The first paper is referenced herein as Gilkes and Chrispeels (1980)     

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     

A precursor of the reserve-protein, phaseolin, is transiently associated with the endoplasmic reticulum of developing Phaseolus vulgaris cotyledons

Developing cotyledons of Phaseolus vulgaris L. were labeled for 30 min with [³H] amino acids, homogenized, and the proteins fractionated on sodium dodecylsulfate (SDS) polyacrylamide gels. Fluorographs of these gels showed that the polypeptides of phaseolin, the major reserve protein of P. vulgaris, were synthesized as precursors which could be distinguished from the polypeptides of mature phaseolin by their slightly lower mobility. When extracts of cotyledons labeled for 45 min with [³H] amino acids were fractionated on isopynic sucrose gradients, radioactive phaseolin banded at the same density (1.14 g cm^-3) as the endoplasmic reticulum (ER)-marker enzyme NADH-cytochrome c reductase. Fractionation in the presence of 3 mM MgCl₂ indicated that the newly-synthesized phaseolin was associated with the rough ER. Pulse-chase experiments showed that phaseolin was transiently associated with the ER, and later accumulated in the protein bodies. Treatment of isolated ER with proteinase K showed that phaseolin polypeptides were degraded only if Triton X-100 was present, indicating that phaseolin was membrane-protected, probably enclosed within the vesicles. ER-associated phaseolin associated to an 18S form at pH 4.5 in the presence of 0.3 M NaCl and 100 mM sodium acetate. The polypeptides of ER-associated phaseolin had a slightly lower mobility on SDS-gels than polypeptides of protein body phaseolin. ER-associated phaseolin had a carbohydrate content of 6.8%, while protein body-derived phaseolin had a carbohydrate content of 6.2%. When cotyledons were labeled simultaneously with [¹⁴C] amino acids and [³H] glucosamine or with [¹⁴C] amino acids and [³H] mannose, the [³H]/[¹⁴C] ratio of ER-derived phaseolin was similar to that of protein body derived phaseolin, indicating that the faster mobility on SDS-gels was not due to the detachment of carbohydrate. Experiments in which the carbohydrate side chains were removed with endoglycosidase H, and the resulting polypeptides subjected to electrophoresis in SDS-gels showed that the differential mobility of the glycopolypeptides of phaseolin resided in their polypeptide chains.     

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     

Immunocytochemical localisation of lectins in cells of Phaseolus vulgaris L. seeds

Antibodies against pure E₄- and L₄-lectins from the seeds of Phaseolus vulgaris L. raised in rabbits were made monospecific by immunoaffinity chromatography on E₄- or L₄-lectin Sepharose 4B columns. Localisation of lectins in bean seeds was investigated by indirect immunofluorescence and by electron microscopy on sections stained with colloidal gold particles coated with monospecific anti-E₄- and anti-L₄-IgG. In parenchyma cells from the cotyledons both E- and L-type lectins were found inside the protein bodies. Apparently the matrix of all protein bodies contained both types of lectins. On the other hand in vascular and in axis cells the two types of lectins were localised in the cytoplasm, outside the protein bodies. Thus these findings suggest different roles for the lectins: in cotyledons this may be a specific form of N storage, while in vascular and axis cells lectins may have a more direct metabolic part to play.     

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     

Control of hemicellulose and pectin synthesis during differentiation of vascular tissue in bean (Phaseolus vulgaris) callus and in bean hypocotyl

Membrane fractions from bean hypocotyl or callus incorporate arabinose from UDP--L-arabinose into arabinan and xylose from UDP--D-xylose into xylan. The control of these syntheses has been studied during xylogenesis in stele and in xylogenesis induced in callus tissue. Induction of arabinan synthetase activity occurs during division and extension growth while that of xylan synthetase occurs subsequently during the period of secondary thickening of the cell wall. The xylan synthetase induction is correlated with the induction of phenylalanine ammonia-lyase and with lignin synthesis.Abbreviations PAL phenylalanine ammonia-lyase - NAA 3-naphthylacetic acid - CMD medium supplemented with 2,4-dichlorophenoxy-acetic acid and coconut milk - IM induction medium - MM maintenance medium - EDTA ethylendiamine tetracetate - TCA trichloroacetic acid - DEAE diethylaminoethyl - TLC thin layer chromatography - UDP uridine diphosphate     

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.     

The effect of solubilisation on the character of an ethylene-binding site from Phaseolus vulgaris L. cotyledons

The ethylene-binding site (EBS) from Phaseolus vulgaris cv. Canadian Wonder cotyledons can be solubilised from 96,000 g pelleted material by Triton X-100 or sodium cholate. Extraction of 96,000 g pellets with acetone, butanol or butanol and ether results in a total loss of ethylene-binding activity. Like the membrane-bound form, the solubilised EBS has an apparent K_D(liquid) of 10^-10 M at a concentration of 32 pmol EBS per gram tissue fresh weight. Propylene and acetylene act as competitive inhibitors, carbon dioxide appears to promote ethylene binding and ethane has no significant effect. The solubilised EBS is completely denatured affect. The solubilised EBS is completely denatured after 10 min at 70°C, by 1 mM mercaptoethanol and 0.1 mM dithiothreitol, but not by trypsin or chymotrypsin. However, solubilisation decreases the rate constant of association from 10³ M^-1 s^-1 to 10¹–10² M^-1 s^-1 and hence does not permit experimental determination of the rate constant of dissociation. The pH optimum for ethylene binding is altered from the range pH 7–10 in the membrane-bound form to the pH range 4–7 in the solubilised form. The EBS appears to be a hydrophobic, intergral membrane protein, which requires a hydrophobic environment to retain its activity. Partitioning of the EBS into polymer phases is determined by the detergent used for solubilisation indicating that when solubilised, the EBS forms a complex with detergent molecules.Abbreviations EBS ethylene-binding site - PEG polyethylene glycol     

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves

Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1 -dimethylurea - OC osmotic concentration - RL red light - TX tentoxin - WL white light We thank Dr. G.E. Templeton, University of Arkansas, Fayetteville, USA, for initially supplying us with TX, and also Dr. Stephen O. Duke, Southern Weend Science Laboratory, Stoneville, Miss., USA, for suggesting this compound for our experiments. We are grateful to Professor E. Ballio for his generous gift of fusicoccin.