Purification and characterization of putrescine synthase from cucumber seedlings. A multifunctional enzyme involved in putrescine biosynthesis

The multifunctional enzyme, putrescine synthase has been purified fromCucumis sativus and characterized. This enzyme harbours agmatine iminohydrolase, ornithine transcarbamylase, putrescine transcarbamylase and carbamate kinase activities, whose concerted action results in agmatine → putrescine conversion. The enzyme resolved into two aggregation forms, enzyme aggregated and enzyme monomer upon electrophoresis at pH 8.3. Evidence has been provided by two-dimensional gel electrophoresis that both enzyme aggregated and enzyme monomer comprise of identical polypeptide chains. Under non-reducing conditions on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, the protein moves as a single 150 KDa polypeptide; however, in the presence of 2-mercaptoethanol on sodium dodecyl sulphate-polyacrylamide gel elec trophoresis, it migrates as 3 polypeptides of molecular weight 48,000, 44,000 and 15,000. The enzyme undergoes age-dependentin vivo proteolytic degradation from a 66 KDa polypeptide (primary translational product), through 48 KDa polypeptide to 44 KDa species and finally to small molecular weight peptides. Preliminary results of this work were presented at Golden Jubilee and Annual General Body Meetings of Society of Biological Chemists (India) and the Second Congress of Asian and Ocean Biochemists (1980) held at Bangalore, 1981,Indian J. Biochem. Biophys.,18, 113.     

Hemocyte surface changes in the migratory grasshopper,Melanoplus sanguinipes in response to wounding and infection withBeauveria bassiana

Surface changes of hemocytes from the migratory grasshopper,Melanoplus sanguinipes (Fab.), following wounding and/or infection withBeauveria bassiana (Bals.) Vuillemin were assessed using fluorescein labelled wheat germ agglutinin (WGA) and concanavalin A (Con A). Binding was observed on outer wall of hemocytes when either Con A or WGA conjugates were tested. After infection there was an increase in the percentage of hemocytes which bound to WGA and which remained so for 24 h. However, after wounding this increase in binding subsided after only 1 h. Furthermore, it was found that the increase in WGA-binding occurred within the first 15 min of wounding or infection. In addition, ConA binding in males only increased 3-fold in fungus-injected insects within 1 h of treatment and remained high for 24 h. These results indicated the presence of mannose (and/or glucose) residues (Con A specificity) and N-acetyl-D-glucosamine residues (wheat germ agglutinin; WGA specificity) on outer wall surfaces of the hemocytes. The implications of these results in the immune response ofM. sanguinipes to infection withB. bassiana are discussed.     

Introduction of Thinopyrum intermedium ssp. trichophorum chromosomes to wheat by trigeneric hybridization involving Triticum,Secale and Thinopyrum genera

Main conclusion

Fluorescence in situ hybridization and molecular markers have confirmed that several chromosomes from Thinopyrum intermedium ssp. trichophorum have been added to a wheat background, which originated from a cross between a wheat– Thinopyrum partial amphiploid and triticale. The lines displayed blue grains and resistance to wheat stripe rust.

Thinopyrum intermedium has been used as a valuable resource for improving the disease resistance and yield potential of wheat. With the aim to transfer novel genetic variation from Th. intermedium species for sustainable wheat breeding, a new trigeneric hybrid was produced by crossing an octoploid wheat–Th. intermedium ssp. trichophorum partial amphiploid with hexaploid triticale. Fluorescence in situ hybridization (FISH) revealed that Thinopyrum chromosomes were transmitted preferably and the number of rye chromosomes tended to decrease gradually in the selfed derivatives of the trigeneric hybrids. Four stable wheat–Th. intermedium chromosome substitution, addition and translocation lines were selected, and a 2J^S addition line, two substitution lines of 4J^S(4B) and 4J(4B), and a small 4J.4B translocation line were identified by FISH and molecular markers. It was revealed that the gene(s) responsible for blue grains may located on the FL0.60–1.00 of long arm of Th. intermedium-derived 4J chromosome. Disease resistance screenings indicated that chromosomes 4J^S and 2J^S appear to enhance the resistance to stripe rust in the adult plant stage. The new germplasm with Th. intermedium introgression shows promise for utilization of Thinopyrum chromosome segments in future wheat improvement.

     

Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians

Xanthomonas campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting.mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A⁺ RNA in rabbit reticulocyte lysate in the presence of ³⁵S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these had disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.     

Expression of beta‐galactosidase and pig leptin gene in vitro by recombinant adenovirus

Abstract

Adenovirus has been used in vivo and in vitro as a vector to carry a foreign gene for gene transfer. Two kinds of replication defective human recombinant adenovirus vectors were used in this study, the first containing β‐galactosidase reporter gene (AdCMVLac‐Z) and the second carrying a gene for porcine leptin gene (AdCMVpLeptin). AdCMVLac‐Z was tested for its ability to transfer DNA into pig kidney and pituitary cells. These cells expressed Lac‐Z transiently 48 hours after the infection. In addition, when the pig kidney cells expressing the Lac‐Z were replated with low density for the formation of colonies from each cell, colonies of blue cells expressing Lac‐Z were observed. These results demonstrate that human recombinant adenovirus can be used as a transducing viral vector for inducing long‐term expression in pig kidney cells. We also constructed a recombinant adenovirus (AdCMVpLeptin) which contained a pig leptin gene for the expression of pig leptin in vitro in the 293 human kidney cell line. 293 cells transfected with AdCMVpLeptin produced both a 15 KDa of a secretory form of porcine leptin and an 18 KDa long form containing signal peptide. Our study demonstrated that the recombinant adenovirus system offers a method for gene transfer and expression in pig cells.     

Spatial and temporal analysis of the local response to wounding

We studied the local response to wounding in Arabidopsis thaliana leaves using a two-step microarray analysis. A microarray containing 3500 cDNA clones was first screened to enrich for genes affected by wounding in the immediate vicinity of the wound (4 h post wounding). 359 non-redundant putative wound responsive genes were then spotted on a smaller wound-response array for detailed analysis of spatial expression (local, adjacent and systemic), timing of expression (0.5, 4, 8, 17 h), and effect of hormone treatments (methyl jasmonate, ethylene and abscisic acid). Our results show that genes that respond early at the site of the wound also respond throughout the plant, with similar kinetics. Early-induced genes which respond systemically encode predominantly signal transduction and regulatory factors (36%), and the expression of many of them is also controlled by methyl jasmonate (about 35% of the 36%). Genes specific to the wound site and the wounded leaf have a slower response to wounding and are mainly metabolic genes. At the wound, many genes of the lignin biosynthesis pathway were induced. In silico analysis of the 5′ promoter regions of genes affected by wounding revealed G-box-related motifs in a significant proportion of the promoters. These results show that the establishment of a systemic response to wounding is a priority for the plant, and that the local response at the wound site is established later. Ethylene and abscisic acid are involved in the local response, regulating repression of photosynthetic genes and expression of drought responsive genes respectively.     

The heterogeneity of the plasma membrane H+-ATPase response to auxin

The sensitivity of the plasma membrane H⁺-ATPase in tobacco was investigated in vitro, both at the proton translocation level and the ATPase level, according to plant development and leaf location. Both activities are stimulated by auxin in all leaves, whatever the plant age and the leaf age. However, the sensitivity to auxin was heterogeneous with respect to plant development and leaf location. In parallel experiments using the same plasma membrane samples, polypepides patterns were investigated by two-dimensional gel electrophoresis and image analysis was used to quantify the relative abundance of 110 peptides. Systematic analysis of the two kinds of data identified 8 polypeptides, the abundance of which changed in a consistent way with the sensitivity, whatever the plant developmental state and leaf location. These unknown polypeptides are proposed as potential markers of the membrane response to auxin.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

Proteomic profiles of thylakoid membranes and changes in response to iron deficiency

The proteomic profile of thylakoid membranes and the changes induced in that proteome by iron deficiency have been studied by using thylakoid preparations from Beta vulgaris plants grown in hydroponics. Two different 2-D electrophoresis approaches have been used to study these proteomes: isoelectrical focusing followed by SDS PAGE (IEF-SDS PAGE) and blue-native polyacrylamide gel electrophoresis followed by SDS PAGE (BN-SDS PAGE). These techniques resolved approximately 110–140 and 40 polypeptides, respectively. Iron deficiency induced significant changes in the thylakoid sugar beet proteome profiles: the relative amounts of electron transfer protein complexes were reduced, whereas those of proteins participating in leaf carbon fixation-linked reactions were increased. A set of polypeptides, which includes several enzymes related to metabolism, was detected in thylakoid preparations from Fe-deficient Beta vulgaris leaves by using BN-SDS PAGE, suggesting that they may be associated with these thylakoids in vivo. The BN-SDS PAGE technique has been proven to be a better method than IEF-SDS PAGE to resolve highly hydrophobic integral membrane proteins from thylakoid preparations, allowing for the identification of complexes and determination of their polypeptidic components.     

Nodule-specific glutamine synthetase is expressed before the onset of nitrogen fixation in Phaseolus vulgaris L.

Glutamine synthetase expression was studied in developing root-nodules of common bean with regard to the time-course of specific activity, antigen accumulation, polypeptide composition and in vitro translation products. This analysis shows that the nodule-specific GS polypeptide (GS-gamma) is detected prior to the nitrogenase acetylene-reducing activity, and that its accumulation together with that of the GS-alpha and GS-beta polypeptides vary with nodule age. GS-gamma is present in ineffective nodules, although in a lower ratio to GS-beta than in wild-type nodules. Comparisons of in vitro translated and in vivo synthesized GS polypeptides suggest no post-translational modifications. The possible factors and mechanisms involved in the regulation of expression of GS polypeptides are discussed.     

Comparative proteome analysis of metabolic proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress

In Central and Southern Italy, where durum wheat represents one of the most widely cultivated crops, grain filling occurs during Spring, a period characterized by sudden increases in temperature. Wheat grain proteins are classified into albumins, globulins, and prolamins. The nonprolamin fractions include proteins with metabolic activity or structural function. In order to investigate the consequences of heat stress on the accumulation of nonprolamin proteins in mature durum wheat kernels, the Italian cultivar Svevo was subjected to two thermal regimes (heat stress versus control). The 2‐D patterns of nonprolamin proteins were monitored to identify polypeptides affected by heat stress during grain fill. This study shows that heat stress alters significantly the durum wheat seed proteome, although the changes range is only between 1.2‐ and 2.2‐fold. This analysis revealed 132 differentially expressed polypeptides, 47 of which were identified by MALDI‐TOF and MALDI‐TOF‐TOF MS and included HSPs, proteins involved in the glycolysis and carbohydrate metabolism, as well as stress‐related proteins. Many of the heat‐induced polypeptides are considered to be allergenic for sensitive individuals.     

Head‐group acylation of monogalactosyldiacylglycerol is a common stress response,and the acyl‐galactose acyl composition varies with the plant species and applied stress

Formation of galactose‐acylated monogalactosyldiacylglycerols has been shown to be induced by leaf homogenization, mechanical wounding, avirulent bacterial infection and thawing after snap‐freezing. Here, lipidomic analysis using mass spectrometry showed that galactose‐acylated monogalactosyldiacylglycerols, formed in wheat (Triticum aestivum) and tomato (Solanum lycopersicum) leaves upon wounding, have acyl‐galactose profiles that differ from those of wounded Arabidopsis thaliana, indicating that different plant species accumulate different acyl‐galactose components in response to the same stress. Additionally, the composition of the acyl‐galactose component of Arabidopsis acMGDG (galactose‐acylated monogalactosyldiacylglycerol) depends on the stress treatment. After sub‐lethal freezing treatment, acMGDG contained mainly non‐oxidized fatty acids esterified to galactose, whereas mostly oxidized fatty acids accumulated on galactose after wounding or bacterial infection. Compositional data are consistent with acMGDG being formed in vivo by transacylation with fatty acids from digalactosyldiacylglycerols. Oxophytodienoic acid, an oxidized fatty acid, was more concentrated on the galactosyl ring of acylated monogalactosyldiacylglycerols than in galactolipids in general. Also, oxidized fatty acid‐containing acylated monogalactosyldiacylglycerols increased cumulatively when wounded Arabidopsis leaves were wounded again. These findings suggest that, in Arabidopsis, the pool of galactose‐acylated monogalactosyldiacylglycerols may serve to sequester oxidized fatty acids during stress responses.     

A Possible Extra-Ovarian Site for Synthesis of Lipovitellin During Vitellogenesis in Artemia sp. (Crustacea; Anostraca)

Summary

Lipovitellin samples, extracted from yolk platelets of cysts, were applied to SDS-PAGE. A female specific antiserum was raised against the high molecular weight apoprotein lipovitellin alpha-1 (LV-α1) of the lipovitellin complex. This anti-LV-α1 was used in the peroxidase-anti-peroxidase staining method with frontal paraffin sections (of 4μm) of whole embedded Artemia. Females were studied during a complete vitellogenic cycle. The presence of exogenous yolk precursors in the fat storage cells of the thoracopods of female Artemia was demonstrated. The amount of the female specific yolk polypeptides and the number of positively stained cells changes during the vitellogenic cycle. In vitro experiments with ³⁵S-radiolabelled methionine show the synthesis of lipovitellin-like substances in the fat storage cells of vitellogenic females.     

Expression of three osmotin-like protein genes in response to osmotic stress and fungal infection in potato

We have characterized three cDNAs encoding osmotin-like proteins from potato (Solanum commersonii) cell cultures. These cDNAs (pA13, pA35, and pA81) have extensive nucleotide identity in the coding regions but low homology in the 3 non-coding sequences, and may encode three isoforms of potato pathogenesis-related (PR) type 5 proteins. Using gene-specific probes, RNA gel blot analyses showed constitutive accumulation of osmotin-like protein mRNAs in cell cultures, leaves, stems, roots and flowers, with high abundance in the roots and mature flowers. Treatments with abscisic acid (ABA), low temperature, and NaCl increased the accumulation of all three mRNAs in S. commersonii cell cultures and plants grown in vitro. Salicylic acid (SA), and wounding resulted in a moderate increase in the levels of pA13 and pA81 but not pA35 mRNAs. Infection with the fungus Phytophthora infestans activated strong and non-systemic expression of all three osmotin-like protein genes. The accumulation of osmotin-like proteins, however, was detected only in P. infestans-infected tissues but not in plants treated with ABA, SA, NaCl, low temperature, or wounding.     

In Vitro Synthesis of Wheat (Triticum aestivum L.) Storage Proteins

Free and membrane-associated polysomes were isolated in approximately equal amounts from endosperm of wheat kernels harvested 20 days after anthesis. The presence of heparin in the homogenizing buffer minimized polysome degradation. Ribonucleic acid from the isolated polysomes, when translated in vitro in a wheat germ system, yielded products ranging in size from about 12,000 to about 80,000 daltons, including at least two polypeptides that co-migrated with seed extract proteins in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The nature of the translation products of free and membrane-associated RNA are distinctly different, with membrane-associated RNA yielding a higher proportion of polypeptides in the size range of 30,000 to 37,000 daltons. Analysis of membrane-associated 3′-terminal polyadenylyl-containing RNA in vitro translation products, by solubility in 70% ethanol and by immunoprecipitation, indicates that the 33,000- to 37,000-dalton polypeptides contain gliadins, and the analysis provides evidence that these proteins are synthesized in association with membranous cell organelles. Gliadin polypeptides synthesized in vitro are larger than authentic gliadins and probably are precursors which, in vivo, undergo modification to yield the smaller final products.     

Genes for tolerance to water and osmotic stress in glycophyte plants

Abstract

Most of the crops are extremely susceptible to environmental stresses, because they have been selected for a high yield performance under optimal growth conditions. Not tolerant plants (glycophytes) respond to changes in the environment with complex mechanisms, involving a network of genes, which are either up- or down-regulated. Some of the genes induced are common between glycophytes and typically tolerant plants, e.g. xerophytes and halophytes, supporting the contention that stress tolerance mechanisms are ubiquitous.

In this paper the changes in gene expression observed in potato cells upon abrupt imposition of water stress compared to the changes induced during a gradual acclimation to the same conditions are reported. Data on the involvement or not of the phytohormone abscisic acid in controlling such changes are also reported. The regulation of specific genes (osmotin, Em) during the stress have been studied in glycophyte plants, such as tomato and wheat.