Nuclear Overhauser effect investigation on GM1 ganglioside containingN-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer)

The conformational properties of the oligosaccharide chain of GM1 ganglioside containingN-glycolyl-neuraminic acid, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-were found to fix the relative orientitation of the three saccharides, while the glycosidic linkage of the terminal -Gal-was found to be quite mobile as the -Gal-(1-3)--GalNAc-disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containingN-acetyl-neuraminic acid and neuraminic acid [1].Abbreviations Ganglioside nomenclature is in accordance with Svennerholm [23] and the IUPAC-IUB Recommendations [24] GM3(Neu5Ac) II³Neu5AcLacCer, -Neu5Ac-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM3(Neu5Gc) II³Neu5GcLacCer, -Neu5Gc-(2-3)--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Ac) II³Neu5AcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu5Gc) II³Neu5GcGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu5Gc-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GM1(Neu) II³NeuGgOse₄Cer, -Gal-(1-3)--GalNAc-(1-4)-[-Neu-(2-3)]--Glc-(1-1)-Cer - GD1a IV³Neu5AcII³Neu5AcGgOse₄Cer, -Neu5Ac-(2-3)--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - GalNAc-GD1a IV⁴GalNAcIV³Neu5AcII³Neu5AcGgOse₄Cer, -GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-3)--GalNAc-(1-4)-[-Neu5Ac-(2-3)]--Gal-(1-4)--Glc-(1-1)-Cer - Neu neuraminic acid - Neu5Ac N-acetyl-neuraminic acid - Neu5Gc N-glycolyl-neuraminic acid - Cer ceramide     

Composition of poly-β-hydroxyalkanoate from Syntrophomonas wolfei grown on unsaturated fatty acid substrates

Poly--hydroxyalkanoate (PHA) from crotonate-grown cultures of Syntrophomonas wolfei contained only the d-isomer of -hydroxybutyrate. The PHA from cultures grown with trans-2-pentenoate or one of several hexenoates as the substrate also contained small amounts (5%) of -hydroxypentanoate or -hydroxyhexanoate, respectively. Thus, some PHA was synthesized without cleavage of the carbon skeleton of the substrate, but the predominant route for PHA synthesis was by the condensation and subsequent reduction of acetyl-coenzyme A (CoA). The ratio of the -hydroxypentanoate to the -hydroxybutyrate in PHA in pentenoate-grown cultures increased immediately after inoculation and then decreased as the amount of the -hydroxybutyrate in PHA increased. The amount of -hydroxypentanoate in the PHA did not markedly change throughout the remainder of growth. These data indicated that the unbroken carbon-chain was used for polymer production only in the early stages of growth and, later, polymer synthesis occurred by the condensation and reduction of acetyl-CoA molecules.     

Cell wall localization of dihydroflavonol-glucoside β-glucosidase in flowers of Petunia hybrida

Limbs of flower buds from Petunia hybrida were investigated for -glucosidase activity with dihydroflavonol-glucosides and 4-methyl-umbelliferyl--D-glucoside as substrates. Dihydroflavonol-glucoside -glucosidase is localized in the cell wall. This activity has an acid pH optimum and is also active toward 4-methyl-umbelliferyl--glucoside. Besides this activity a neutral -glucosidase is present. This activity is soluble and is not active toward dihydroflavonol-glucosides. Using starch gel electrophoresis it was shown that no difference in -glucosidase activity is present between mutants able to convert dihydroflavonols into anthocyanins and mutants accumulating dihydroflavonol-glucosides. It is concluded that -glucosidase activity is not involved in anthocyanin synthesis.Abbreviations 4MU--glc 4-methylumbelliferyl--D-glucopyranoside - dHQ-7-g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside - dHM-4-g dihydromyricetin-4-glucoside Deceased     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Purification and characterization of aspartate aminotransferase from developing embryos of the camel tick Hyalomma dromedarii

Aspartate transaminase (AST) activity in the camel tick Hyalomma dromedarii was followed throughout embryogenesis. During purification of AST to homogeneity, ion exchange chromatography lead to four separate forms (termed I, II, III and IV). AST II with the highest specific activity was pure after chromatography on Sephacryl S-300. The molecular mass of AST II was 52KDa for the native enzyme, composed of one subunit of 50KDa. AST II had a Km value of 0.67mM for -ketoglutarate and 15.1mM for aspartate. AST II had a pH optimum of 7.5 with heat stability up to 50°C for 15min. The enzyme was activated by MnCl₂, and inhibited by CaCl₂, MgCl₂, NiCl₂, and ZnCl₂.     

Primary structure of the marmoset (Saguinus fuscicollis) hemoglobin. I. Use of tryptic maleylated peptides in the solubilization and sequence elucidation of the α- and β-chains

The primary structure of adult marmoset hemoglobin has been determined. The - and -chains of HbA were separated on a CM23 column in 8 M urea using a sodium phosphate gradient. Tryptic digests of the - and -chains were fractionated on a Dowex 50W-X2 column using a pH and pyridine acetate gradient. Large peptide fragments were obtained by the cyanogen bromide cleavage of the - and -chains, as well as by tryptic digestion of the maleylated - and -chains. The sequence was derived from the amino acid compositions and sequences of the individual tryptic peptide, automated sequence determination of intact - and -chains, as well as automated sequence determination of cyanogen bromide fragments and tryptic maleylated peptides derived from the - and -chains. The complete structure of marmoset adult hemoglobin is closely homologous to that of other primate hemoglobins. The sequence of the marmoset -chain differs from the -chain of human HbA at positions 8, 19, 23, 68, and 116. The -chain from marmoset HbA differs from the -chain of human HbA at positions 5, 13, 21, 50, 87, and 125.This work was supported in part by funds from a Physicians' Medical Education and Research Foundation Grant of the University of Tennessee Memorial Research Hospital and by NIH General Research Support Grant FR-5541 to the institution.     

Observations on the fine structure of the cercaria of Notocotylus attenuatus and formation of the cyst wall of the metacercaria

Summary The dorsal tegument of the mature cercaria of Notocotylus attenuatus is a syncytial, cytoplasmic layer, containing two types of secretory granule which are identifiable ultrastructurally. The type 1 secretory bodies are electron lucid, whereas most type 2 granules have a banded appearance. The ventral tegument contains granules which are secreted from the type 3 cells; the type 3 granules are membrane bound, electron dense, and consist of both an amorphous and a finely striated zone. The type 4 cells mainly contain cigar-shaped granules consisting of an amorphous core surrounded by concentric striations. The granules exhibit structural variability in shape and content. The type 4 cells undergo a cellular migration to the tegument during encystment. The structure of the posterior-lateral glands and mode of secretion of the granules are described. Possible functions of microtubules are discussed for each cell type. Details of some secretory processes involved in the formation of the hemispherical cyst wall are described. The layers of the cyst wall may be related to the granular contents of the various parenchymal cells of the cercaria. The tegument of the metacercaria originates primarily from the cytoplasm of the type 1, type 2, type 3 and type 4 cells.     

Involvement of Erks activation in cadmium‐induced AP‐1 transactivation in vitro and in vivo

Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category 1 carcinogen. Cadmium-induced upregulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP1 activity by cadmium appears to involve activation of Erks, since the induction of AP1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP1 in AP1luciferase report transgenic mice. Considering the role of AP1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmiuminduced carcinogenesis.     

Identification of a complete set of isogenic wheat/rye D-genome substitution lines by means of Giemsa C-banding

Summary A complete set of isogenic wheat/rye D-genome substitutions were produced by crossing an inbred line of spring rye Secale cereale L. cv. Prolific to a tetraploid wheat, the A-and B-genomes of which had previously been extracted from hexaploid wheat, Triticum aestivum L. em Thell. cv. Thatcher. After chromosome doubling, the derived hexaploid triticale (x Triticosecale Wittmack) was backcrossed to 6x Thatcher and selection for wheat/rye substitution lines was carried out in BCF₃ to BCF₆ families by using Giemsa C-banding. Five fertile disomic wheat/rye D-genome substitution lines were obtained and their chromosomal constitution was determined to be 1D/1R, 2D/2R, 7D/4R, 6D/6R, 7D/7R. The two remaining 3R and 5R substitutions are at the moment in a monosomic condition. Another 1D/7R substitution was detected but this plant was very weak and sterile, indicating that only substitutions between homoeologous chromosomes result in fertile, vigorous plants. Furthermore, many rye telocentrics as well as rye-rye and rye-wheat translocations were selected. Since all lines selected in this program share the same genetic background of Thatcher wheat, genetic heterogeneity is excluded. The material is very useful, therefore, for analyzing the effects of different rye chromosomes or chromosome segments in an otherwise homozygous background.Contribution No. 797     

Nuclear and cytoplasmic gene control of resistance to loose smut (Ustilago tritici (Pers.) Rostr.) in wheat (Triticum aestivum L.)

Summary Using disomic chromosome substitution lines based on the susceptible wheat cultivar Chinese Spring, loose smut resistance of wheat cultivars Hope and Thatcher was shown to be conferred in each case by a single dominant major gene carried on chromosome 7 A (Hope) or 7 B (Thatcher). Partial resistance was determined by genes on an additional eight Hope or seven Thatcher chromosomes, and similarities were evident between the partial resistance genotypes ofHope and Thatcher. Chinese Spring exhibited a mean infection value of approximately 50%, indicating a significant level of partial resistance, which was found to be due, in part, to genes on the homoeologous chromosome arms 1 As, 1 Es and 1 Ds, and to cytoplasmic genes. Substitution of the Chinese Spring nucleus into the cytoplasm of Aegilops squarrosa, Ae. variabilis or Ae. mutica resulted in increased susceptibility to Ustilago tritici. Several alloplasmic lines of the resistant wheat cultivars Selkirk and Chris exhibited race-specific susceptibility to U. tritici.     

The relation of proton motive force,adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction inBacillus licheniformis under aerobic growth conditions

The magnitude of the proton motive force (p) and its constituents, the electrical () and chemical potential (-ZpH), were established for chemostat cultures of a protease-producing, relaxed (rel ^–) variant and a not protease-producing, stringent (rel ⁺) variant of an industrial strain ofBacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of p with the specific growth rate was found. The calculated intracellular pH (pH_in) was not constant but inversely related to . This change in pH_in might be related to regulatory functions of metabolism but a regulatory role for pH_in itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low and an increased value at higher . For both limitations, the ATP/ADP ratio was directly related to .The phosphorylation potential (G'p) was invariant with . From the values for G'p and p, a variable H⁺/ATP-stoichiometry was inferred: H⁺/ATP=1.83+0.52µ, so that at a given H⁺/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for =0 to _max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.Abbreviations CCCP carbonylcyanide-p-trichloromethoxyphenylhydrazone - DW dry weight of biomass - F Faraday's constant, 96.6 J/(mV × mol) - Fo chemostat outflow-rate (ml/h) - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - G'p phosphorylation potential, the Gibbs energy change for ATP-synthesis from ADP and P_i - G'⁰p standard Gibbs energy change at specified conditions - H⁺/ATP number of protons translocated through - ATP synthase in synthesis of one ATP - H⁺/O protons translocated during transfer of 2 electrons from substrate to oxygen - specific growth rate (1/h) - _H+ transmembrane electrochemical proton potential, J/mol - M_b molar weight (147.6 g/mol) of bacteria with general cell formula C_6.0H_10.8O_3.0N_1.2 - pH_out,in extracellular, intracellular pH - P_i (intracellular) inorganic phosphate - p proton motive force, mV - pH transmembrane pH-difference - transmembrane electrical potential, mV - P/O number of ADP phosphorylated to ATP upon reduction of one O^2– to H₂O by two electrons transferred through the electron transfer chain - P/O (H⁺/O) × (H⁺/ATP)^–1 - P/O_F, P/O_N P/O with the two electrons donated by resp. (NADH + H⁺) and FADH - q specific rate of consumption or production (mol/g DW × h) - rel ⁺,rel ^– stringent, relaxed genotype - R universal gas constant, 8.36 J/(mol × degree) - T absolute temperature - TPMP⁺ triphenylmethylphosphonium ion - TPP⁺ tetraphenyl phosphonium ion - Y growth yield, g DW/mol - Z conversion constant=61.8 mV for 310 K (37 °C) - ZpH transmembrane proton potential or chemical potential, mV     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.     

Subtle behavioral variation in wild chimpanzees, with special reference to Imanishi’s concept of kaluchua

Here we consider the concept of kaluchua (a word adopted from the English culture) in group-living animals developed by Imanishi in the 1950s. He distinguished it from bunka (the Japanese equivalent to the English culture) because he thought that bunka had strong connotations of noble and intellectual human-like activities. Although he did not rigidly define kaluchua, his original concept of kaluchua was much broader than bunka and represented non-hereditary, acquired behavior that was acknowledged socially. However, instead of social life, complex feeding skills have often formed the central topic in the current studies of animal culture. In order to provide evidence that more subtle behavioral variations exist among wild chimpanzee (Pan troglodytes) populations, we directly compared the behaviors of two well-habituated chimpanzee groups, at Bossou and Mahale. During a 2-month stay at Bossou, M.N. (the first author) saw several behavioral patterns that were absent or rare at Mahale. Two of them, mutual genital touch and heel tap were probably customary for mature females and for mature males, respectively. Index to palm and sputter are still open to question. These subtle patterns occurred more often than tool use during the study period, suggesting that rarity is not the main reason for their being ignored. Unlike tool use, some cultural behavioral patterns do not seem to require complex skills or intellectual processes, and sometimes it is hard to explain the existence of such behaviors only in terms of function.     

Morphology and structural organization of spiracles in femaleArgas (Persicargas) walkerae (Acari: Argasidae)

Scanning electron microscopical investigations of fractures and corrosion casts of the spiracles from femaleA. walkerae ticks revealed a four-part structure, consisting of spiracular plate, ostium and macula forming the external closure, followed by the subostial space and the vestibulum of regulable volume, as well as the atrial chamber as the innermost part from which the main tracheal trunks originate. On the average, the spiracular plate was 158 m long and 188 m at the broadest width. It consisted of a thin, highly perforated external and a thick internal layer, which enclosed the interpedicellar space with numerous stout pedicels. In its posterior region, the spiracular plate was covered by the macula, which was up to 80 m in length and 110 m in width. The interpedicellar cavity opened into the subostial space measuring 95.5 m in length and 159.6m in width, which proceeded into the 112-m long vestibulum. The roof of the vestibulum was flexible and could be everted and inverted. Inverted, the roof formed a quadratic bulge with numerous deep cuticular folds, which confined the lumen of the vestibulum either partially or completely. In corrosion casts, the roof was everted to a length of up to 89.3 m. In the posterior part of the vestibulum, as well as in the initial fourth of the artrial chamber, numerous anvil-, cone-or drop-like cuticular projections were arranged in wedge-like fashion. The atrial chamber was almost spherical with a diameter of 138.4 m. Five main tracheal trunks of different luminal diameter as well as numerous channels opened into the atrial chamber.     

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

Summary Studies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cell's long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a marking pin-like configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 M caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 M, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.Abbreviations APM amiprophos-methyl - CD cytochalasin D - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - DMSO dimethyl sulfoxide - Rh-Ph rhodamine-phalloidin     

In vitro binding of riboflavin to subcellular particles from maize coleoptiles and Cucurbita hypocotyls

Saturable and reversible in vitro binding of [¹⁴C]riboflavin was found to occur on subcellular, sedimentable particles from maize coleoptiles and Cucurbita hypocotyls. The K_D was ca. 6 M, the pH optimum was near 6.0, and the number of binding sites amounted to 0.1–0.5 M on a fresh-weight basis. When the reducing agent dithionite was present, riboflavin binding increased-the K_D was 2.5 M, and the pH optimum above 8.0. The binding was specific: flavin mononucleotide (FMN) and flavin adenosine-dinucleotide (FAD) bound less tightly to these sites than riboflavin and another major soluble flavin, the previously described riboflavin-analog FX, occurring in grass coleoptiles. These flavin-binding sites were localized on vesicles derived from plasmalemma and endoplasmic reticulum by analyzing sucrose and metrizamide density gradients and marker enzymes.Abbreviations CCO cytochrome-c oxidase - CCR NADH-cytochrome-c oxidoreductase - ER endoplasmic reticulum - FAD flavin-adenosinedinucleotide - FMN flavin mononucleotide - MOPS N-morpholino-3-propansulfonic acid - NADH reduced -nicotinamide dinucleotide - nKP n thousand times g pellet - NPA l-naphthylphthalamic acid - PM plasma membrane, plasmalemma - RBF riboflavin - IAA indoleacetic acid - BA benzoic acid     

Glycerolipid-fatty-acid desaturase deficiencies in chloroplasts from fruits of Capsicum annuum L.

Chloroplasts from fruits and leaves of Capsicum annuum cv. Bell Tower were purified on sucrose gradients, and the lipids were separated by column and thin-layer chromatography. The glycerolipids mono- and digalactosyldiacylglycerol (MGDG, DGDG), sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol (PG) were quantified, and the fatty-acid composition at the 1 and 2 positions of the glycerol moiety (sn-1 and sn-2) was determined after hydrolysis with position-specific lipases. In fruit chloroplasts, ³-trans hexadecenoate (trans-3-161) was absent and replaced by palmitate (160) at sn-2 of PG, and ^7,10,13-hexadecatrienoate (163) at sn-2 of MGDG was greatly reduced and largely replaced by linoleate (182). The ratio of 182 to linolenate (183) was consistently greater in glycerolipids from fruit compared with leaf chloroplasts. The lower percentage of C-16 fatty acids at sn-2 indicated that prokaryotic molecular species were reduced by 15% in DGDG, 40% in SQDG, and 90% in MGDG, in fruit compared with leaf chloroplasts. The MGDGDGDG ratios in fruit and leaf chloroplasts were 1.21 and 2.21, respectively. Taken together, the data indicate that chloroplasts in Capsicum fruit are deficient in three desaturases: those that convert 1) 160 to ³-trans-161 at sn-2 of PG, 2) 160 to ⁷–cis-161 at sn-2 of MGDG, and 3) 182 to 183 at both sn-1 and sn-2 of various chloroplast glycerolipids.Abbreviations Chl chlorophyll - DGDG digalactosyldiacylglycerol - FS free sterol - GL galactolipid - MGDG monogalactosyldiacylglycerol - PE phosphatidyl ethanolamine - PG phosphatidylglycerol - PL phospholipid - SQDG sulfoquinovosyldiacylglycerol We are grateful to Dr. Roger Calza for providing us with the tobacco gt11 cDNA expression library and to Dr. Eric Huttner for his advice throughout the screening procedure. We also wish to thank M. Gosse for his assistance in growing and maintaining our plants. T.W.B. was supported by a BAP research grant from the Commission of the European Communities.     

Photosynthesis of isolated chloroplasts and protoplasts under osmotic stress

1. Isolated intact spinach chloroplasts respond to changes of the sorbitol concentration of the suspending medium as near-perfect osmometers within a large range of osmotic potentials. Under isotonic conditions (=9–10 bar), their average osmotic volume is 24 m³ and the total volume 36 m³. The osmotic volume can be increased to 63 m³ by lowering the sorbitol concentration until a critical osmotic potential of =4 bar is reached. Below that value chloroplasts rupture. Between 10 bar and 4 bar, volume changes are reversible. 2. Increasing the chloroplast volume above 24 m³ causes inhibition of photosynthesis, with 50% inhibition occurring at an osmotic potential of =5–6 bar. This corresponds to an osmotic volume of 45–55 m³. Depending on the duration of hypotonic treatment, inhibition of photosynthesis is more or less reversible. 3. Between 4 and 10 bar, the chloroplast envelope exhibits a very low permeability for ferricyanide, many metabolites, and soluble stroma proteins. 4. Electron transport is not inhibited by swelling of chloroplasts. Also, the ATP/ADP-ratio remains unchanged. 5. The solute concentration in the chloroplasts appears to be optimal for photosynthesis at 10 bar. Increasing the chloroplast volume causes inhibition of photosynthesis by dilution effects.     

Homologues of a vacuolar processing enzyme that are expressed in different organs in Arabidopsis thaliana

Vacuolar processing enzymes (VPEs) are responsible for the maturation of seed proteins. These processing enzymes belong to a novel group of cysteine proteinases with molecular masses of 37 to 39 kDa. We isolated two genes of VPEs from a genomic library of Arabidopsis. The gene products were designated -VPE and -VPE, and they were 56% identical in terms of amino acid sequence. The amino acid sequences of -VPE and -VPE were also 55% and 67% identical to that of castor bean VPE, respectively. The gene for -VPE had 7 introns, while that of -VPE had 8 introns. Northern blot analysis revealed that -VPE is expressed in rosette leaves, cauline leaves and stems of Arabidopsis, while -VPE is predominantly expressed in the flowers and buds. Neither -VPE nor -VPE is expressed in the siliques. This result strongly suggests that the isolated genes encode isozymes of VPE that are specific to vegetative organs.