Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.     

Isolation and characterization of high-mobility-group proteins from maize

Chromosomal nonhistone high-mobility-group (HMG) proteins were purified from nuclei of maize (Zea mays L. cv. A619) endosperm and leaf tissue. Tissuespecific differences were observed in their polypeptide patterns, in in-vitro phosphorylation experiments with a casein-kinase type II, and by Western blot analysis with antisera against different HMG proteins. Gelfiltration chromatography demonstrated that maize HMG proteins occur as monomers. By measuring the capacity of the HMG proteins to bind to the 5 flanking region of a zein gene, the sensitivity of the proteins to different temperatures, salt concentrations and pH values was determined.Abbreviations EMSA electrophoretic-mobility-shift assay - FPLC fast protein liquid chromatography - HMG high-mobility group - kDa kilodaltons - PVDF polyvinylidenedifluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We would like to thank Mrs. E. Brutzer for excellent technical assistance. We are indebted to Mrs. M. Strecker and Dr. W. Bessler of the Institut für Immunbiologie, Freiburg, FRG, for the preparation of antisera and we gratefully acknowledge helpful discussions with Drs. T. Quayle, R. Grimm and U. Müller of this institute. This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie.     

Isolation and identification of mRNA for the high-molecular-weight storage proteins of wheat endosperm

The prolamin storage proteins of the wheat endosperm contain a sub-class of high-molecular-weight (HMW) polypeptides which have been implicated in determining breadmaking quality. Membrane-bound polysomes isolated from developing wheat endosperms contain mRNA for these HMW components. Although unfractionated polyadenylated RNA derived from the polysomes did not direct the synthesis of these components in an in-vitro wheat-germ system, it did when incubated with a rabbit reticulocyte lysate system. Identification of the translation products as HMW prolamins was based on their large incorporation of [³H]leucine and [³H]glycine relative to [³H]lysine, their mobility on polyacrylamide-gel electrophoresis and the observation that the changes of mobility in response to change in wheat genotype were the same as those observed for the authentic protein. The mRNA was fractionated by electrophoresis and density-gradient centrifugation. The mRNA for the HMW prolamins was found to have a relative molecular mass of about 1.6·10⁶.Abbreviations HMW high molecular weight - PAGE polyacrylamide-gel electrophoresis - poly(A)⁺RNA polyadenylated RNA - SDS sodium dodecyl sulphate     

Growth responses of rice seedlings to triacontanol in light and dark

Triacontanol, a 30-carbon primary alcohol, applied in nutrient culture solutions to rice (Oryza sativa L.) seedlings at 2.3×10^-8 M (10 g/l), caused an increase in dry weight and leaf area of the whole plants. The response could be observed as early as 3 h of treatment. It was observed at relatively high and low light intensities as well as in the dark where control plants lost but triacontanol-treated plants gained in dry weight. The dry weight gain in the dark was, however, eliminated by removing CO₂ from the atmosphere. Triacontanol-treated plants also increased their content of Kjeldahl-N and contained 30% more total N per plant than controls after 6 h in the dark.     

Immunochemical studies on the role of the Golgi complex in protein-body formation in rice seeds

Antibodies raised against purified glutelins and prolamines were employed as probes to study the cellular routes by which these proteins are deposited into protein bodies of rice (Oryza sativa L.) endosperm. Three morphologically distinct protein bodies, large spherical, small spherical, and irregularly-shaped, were observed, in agreement with existing reports. Immunocytochemical studies showed the presence of glutelins in the irregularly-shaped protein bodies while the prolamines were found in both the large and small spherical protein bodies. Both the large and small spherical protein bodies, distinguishable by electron density and gold-labeling patterns, appear to be formed by direct deposition of the newly formed proteins into the lumen of the rough endoplasmic reticulum (ER). In contrast, glutelin protein bodies are formed via the Golgi apparatus. Small electron-lucent vesicles are often found at one side of the Golgi. Electron-dense vesicles, whose contents are labeled by glutelin antibody-gold particles, are commonly observed at the distal side of the Golgi apparatus and fuse to form the irregularly shaped protein bodies in endosperm cells. These observations indicate that the transport of rice glutelins from their site of synthesis, the ER, to the site of deposition, the protein bodies, is mediated by the Golgi apparatus.Abbreviations BSA bovine serum albumin - Da dalton - DAF days after flowering - ER endoplasmic reticulum - GL irregularly shaped - L large spherical - S small spherical (protein bodies) - PBS phosphate-buffered saline - PTA phosphotungstic acid     

Circadian changes in protein-synthesis rate and protein phosphorylation in cell-free extracts of Gonyaulax polyedra

The polysomal pattern of the dinoflagellate Gonyaulax polyedra, cultured under constant conditions, demonstrates a circadian rhythm. The relative amount of polysomes increases during the phase corresponding to the previous night period (=subjective night phase) when the rate of protein synthesis reaches its maximum (Cornelius et al., 1985, Planta 160, 365–370). Cell-free extracts were isolated at different circadian phase. The rate of protein synthesis in the extracts changed rhythmically in the same manner as the rate of protein synthesis in vivo. Substances in the postribosomal supernatants influenced the protein-synthesis rate of the cell-free system, depending on the phase when they were isolated: night factors stimulated protein synthesis in day extracts whereas day factors inhibited protein synthesis in night extracts. These effects were abolished by heating the postribosomal supernatant. In-vitro phosphorylation in parallel probes showed changes in the pattern of phosphorylated proteins. Phosphorylation of one of the proteins (95 kDa) was decreased after addition of night factor(s) and increased after addition of day factor(s). Cyclic-AMP enhanced the rates of protein synthesis and phosphorylation in the day extracts.Abbreviations cAMP cyclic-AMP - CT circadian time - D (N) subjective day (night)phase     

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.     

Purification and characterization of antifreeze proteins from larvae of the beetle Dendroides canadensis

Summary Four antifreeze proteins (AFPs) were purified from larvae of the beetle Dendroides canadensis. The AFPs are similar in amino acid compositions, having high contents of hydrophilic amino acids (45–55 mol%) and cysteine (16 mol% Cys). Approximately half of the Cys residues form disulfide bridges, and both the disulfide bridges and free sulfhydryls are essential for activity. The N-terminals of the AFPs are blocked. The pH optimum of the AFPs is 7.8, but major loss of activity occurred only at very high pH (12.0). The detergents SDS and Triton X-100 did not inactivate the AFPs. Circular dichroism spectra indicate the presence of both and secondary structures in the AFPs, in addition to a large random structure component.Abbreviations AFP antifreeze protein - CD circular dichroism - DTT dithiothreitol - HPLC high pressure liquid chromatography - PAGE polyacrylamide gel electrophoresis - PAS periodic acid Schiff - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid     

Influence of low irradiance on chloroplast proteins and photosystem activities in rice cultivars

In six rice cultivars the relative amounts of 55, 26–28 and 15 kDa polypeptides were mostly lower in plants grown for 15 d under low irradiance than in plants grown under saturating irradiance. This decline in the polypeptides, especially in those related to light-harvesting chlorophyll proteins, was accompanied with a decrease in the whole chain electron transport and the photosystem 2 activity.     

Influence of culture conditions and virulence plasmids on expression of immunoglobulin-binding proteins of Yersinia pseudotuberculosis

The influence of culture conditions and plasmids on immunoglobulin (Ig)-binding activity of two isogenic strains of Yersinia pseudotuberculosis (plasmid-free strain 48(-)82(-) and strain 48(+)82(+) bearing plasmids pYV48 and pVM82) was studied. The highest activity was observed in the bacteria grown on glucose-containing liquid medium in the stationary growth phase. The Ig-binding activity of the bacteria cultured on the liquid medium at pH 6.0 was about 1.5-fold higher than that of the bacteria grown at pH 7.2. Expression of the Ig-binding proteins (IBPs) was most influenced by temperature of cultivation. The IBP biosynthesis was activated in the bacteria grown at 4 degrees C and markedly decreased in those grown at 37 degrees C. The Ig-binding activity of lysates from the bacteria was caused by proteins with molecular weights of 7-20 kD. The activities of the plasmid-free and plasmid-bearing Y. pseudotuberculosis strains (48(-)82(-) and 48(+)82(+), respectively) were analyzed, and the plasmids were shown to have no effect on the IBP expression and biosynthesis, which seemed to be determined by chromosomal genes.     

Mutants for rice storage proteins

Summary To obtain genetic materials to breed qualitatively improved rice storage proteins, we screened about 3,000 mutant lines induced by the treatment of rice fertilized egg cell with N-methyl-N-nitrosourea (MNU). The screening was performed by comparing the profiles of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with that of the original variety, Kinmaze, especially focussing on the changes in polypeptides present in two kinds of protein bodies, PB-I and PB-II. We selected 17 mutant lines and classified them into 4 types on the basis of variations of the relative contents of the polypeptides. Determination of extracted protein in the starchy endosperm of the mutants revealed changes in the content of prolamin and glutelin but not globulin. In some mutants there was marked accumulation of 57 kDa polypeptide concomitant with the remarkable reduction of glutelin subunits. Treatment of the fertilized egg cell with MNU was found to be an effective method to induce mutations for storage proteins in protein bodies of rice.     

Purification and characterization of a novel ribosome-inactivating protein from seeds of Trichosanthes kirilowii Maxim

A novel ribosome-inactivating protein, designated Trichosanthrip, was purified from mature seeds of Trichosanthes kirilowii Maxim by cation-exchange and gel-filtration chromatography. Trichosanthrip migrated as a single band in SDS–PAGE, with an apparent molecular mass of 13 kDa. The molecular mass of Trichosanthrip was 10,964.617 Da as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Trichosanthrip showed N-glycosidase activity on 28 S rRNA and strongly inhibited cell-free protein synthesis, with an IC₅₀ of 1.6 ng/ml. Liquid chromatography–tandem mass spectrometry showed that Trichosanthrip was a novel protein with similar sequence to other proteins present in members of the Cucurbitaceae.     

Regulation of growth in stem sections of deep-water rice

Submergence in water greatly stimulates internodal elongation in excised stem sections of deep-water rice (Oryza sativa L. cv. Habiganj Aman II) and inhibits growth of leaf blades and leaf sheaths. The highest rates of internodal growth have been observed in continuous light. Very little growth occurs in submerged sections kept in darkness or incubated under N₂ in the light. The effect of submergence on the growth of deep-water rice is, at least in part, mediated by C₂H₄, which accumulates in the air spaces of submerged sections. This accumulation results from increased C₂H₄ synthesis in the internodes of submerged sections and reduced diffusion of C₂H₄ from the tissue into the water. Increased C₂H₄ levels accelerate internodal elongation and inhibit the growth of leaves. Compounds capable of changing the rate of C₂H₄ synthesis, namely aminoethoxyvinylglycine, an inhibitor of C₂H₄ synthesis, and 1-aminocyclopropane-1-carboxylic acid, the immediate, precursor of C₂H₄, have opposite effects on growth of internodes and leaves. The enhancement of internodal elongation by C₂H₄ is particularly pronounced in an atmosphere of high CO₂ and low O₂. The increase in C₂H₄ synthesis in internodes of submerged sections is primarily triggered by reduced atmospheric concentrations of O₂. The rate of C₂H₄ evolution by internodes isolated from stem sections and incubated in an atmosphere of low O₂ is up to four times greater than that of isolated internodes incubated in air. In contrast, C₂H₄ evolution from the leaves is reduced under hypoxic conditions. The effect of submergence on growth of stem sections of deep-water rice can be mimicked by exposing non-submerged sections to a gas mixture which is similar to the gaseous atmosphere in the internodal lacunae of submerged sections, namely 3% O₂, 6% CO₂, 91% N₂ (by vol.) and 1 l l^-1 C₂H₄. Our results indicate that growth responses obtained with isolated rice stem sections are similar to those of intact deep-water rice plants.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine     

Purification and characterization of chitin-binding proteins from the hemolymph of sweet potato hornworm, Agrius convolvuli

Three chitin-binding proteins (CBPs: CBP9, CBP15, CBP66) were identified from the larval hemolymph of sweet potato hornworm, Agrius convolvuli.Two (CBP9 and CBP15) of them have been isolated and purified by gel filtration (Superdex HR 75), cation-exchange chromatography (Mono S), and reverse-phase chromatography (μRPC PC 2.1/3). In experiments to detect CBPs in hemolymph, we examined whether ionic strength and existence of bovine serum albumin in the incubation solution influenced binding affinity of CBPs to chitin. The N-terminal sequences of three CBPs were determined by the automated Edman degradation and showed the sequence homology in basic local alignment search tool search. CBP15 and CBP66 were quite similar to lysozymes and bovine serum albumins, respectively. In contrast, CBP9 is not similar to any other known protein, as judged from databank comparisons. Therefore, we concluded that CBP9 is a novel protein with binding capacity to chitin that is a component of the fungal cell wall. CBP9 has no antibacterial activity against Escherichia coli and Micrococcus luteus, and also showed negative response in hemagglutination assay. CBP9 is confirmed as a monomer with a molecular mass of 9.14 kDa by electron spray ionization and matrix-assisted laser desorption ionization mass spectrometry.     

Purification and characterization of barley-aleurone xylanase

Xylanase (-1,4-D-xylan xylanohydrolase; EC 3.2.1.8) from aleurone layers of barley (Hordeum vulgare L. cv. Himalaya) was purified and characterized. Purification was by preparative isoelectric focusing and a Sephadex G-200 column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight (Mr)=34000 daltons. The isoelectric point of the enzyme was 4.6. The enzyme had maximum activity on xylan at pH 5.5 and at 35° C. It was most stable between pH 5 and 6 and at temperatures between 0 and 4° C. The K_m was 0.86 mg xylan·ml^-1.Abbreviations GA₃ gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Drosophila hemolymph proteins: Purification,characterization, and genetic mapping of larval serum protein 2 in D. melanogaster

Three of the major protein species present in the hemolymph of Drosophila melanogaster larvae just prior to pupation are absent from second instar larvae but accumulate rapidly during the third instar. This article describes the purification and characterization of one of these, larval serum protein (LSP) 2, using an immunological assay. It is a homohexamer of molecular weight about 450,000, with a polypeptide molecular weight of 78,000–83,000. Fast and slow electrophoretic variants of this protein map between the markers vin and gs, at 36–37 on chromosome 3.This work was partially supported by M.R.C. Research Studentships to J.W. and M.E.A.     

Immunological characterization of chlorophyll a/b-binding proteins of barley thylakoids

Monoclonal antibodies have been raised against the light-harvesting chlorophyll a/b-binding proteins of photosystem I (LHCI) using a photosystem (PS) I preparation (PSI-200) wild-type from barley (Hordeum vulgare L. cv. Svaløf's Bonus) as the antigen. These antibodies cross-reacted with a minor light-harvesting chlorophyll a/b-protein of PSII (Chla/b-P1=CP29), but not with the major one, LHCII (=Chla/b-P2^**). Similarly, a monoclonal antibody to Chla/b-P1, elicited by a PSII preparation as the antigen, cross-reacted with LHCI, but not LHCII. This explains why an antigen consisting of LHCII, free of LHCI, but contaminated with Chla/b-P1, can elicit antibodies which cross-react with LHCI. Immunoblot assays showed that LHCI and Chla/b-P1 have at least two epitopes in common. Immunogold labelling of thin-sectioned wild-type thylakoids confirmed a preferential localisation of Chla/b-P1 in grana partition membranes and LHCI in stroma lamellae. The presence of LHCI was demonstrated in barley mutants lacking the PSI reaction centre (viridis-zb ⁶³) and chlorophyll b (chlorina-f2), and was correlated with the presence of long-wavelength (730 nm) fluorescence emission at 77 K. The mutant viridis-k ²³, which has a 77 K long-wavelength fluorescence peak at 720 nm, was shown by immune-blot assay to lack LHCI, although Chla/b-P1 was present.Abbreviations Chl-P chlorophyll-protein - CM Carlsberg Monoclonal - Da dalton - LHC light-harvesting complex - PAGE polyacrylamide gel electrophoresis - PSI, II photosystem I, II - PSI-200 PSI containing LHCI polypeptides - SDS sodium dodecyl sulphate