Cotton-fiber germin-like protein. II: Immunolocalization,purification, and functional analysis

Cotton (Gossypium hirsutum L.) contains a germin-like protein (GLP), GhGLP1, that shows tissue-specific accumulation in fiber. The fiber GLP is an oligomeric, glycosylated protein with a subunit size of approximately 25.5 kDa. Accumulation of GhGLP1 occurs during the period of fiber elongation [4–14 days post-anthesis (DPA)]. During early phases of fiber development (2–4 DPA), GhGLP1 localizes to cytoplasmic vesicles as shown by confocal immunofluorescent microscopy. In slightly older fibers (7–10 DPA), GhGLP1 localizes to the apoplast. In other plants, germins and GLPs have been reported to have enzymatic activities including oxalate oxidase (OxO), superoxide dismutase, and ADP-glucose pyrophosphatase. Cotton fiber extracts did not contain OxO activity, nor did intact fibers stain for OxO activity. A four-step purification protocol involving ammonium sulfate precipitation of a 1.0 M NaCl extract, ion-exchange chromatography on DEAE-Trisacryl M, lectin-affinity chromatography, and gel filtration chromatography resulted in electrophoretically pure GhGLP1. While 1.0 M NaCl extracts from 10–14 DPA fiber contained superoxide dismutase and phosphodiesterase activities, GhGLP1 could be separated from both enzyme activities by the purification protocol. Although a GLP accumulates in the cotton fiber apoplast during cell elongation, the function of this protein in fiber growth and development remains unknown.Abbreviations ABP Auxin binding protein - AGPPase ADP-Glucose pyrophosphatase/phosphodiesterase - bis-PNPP Bis-p-nitrophenol phosphate - ConA Concanavalin A - DOA Day of anthesis - DPA Days post-anthesis - GLP Germin-like protein - Mn-SOD Manganese superoxide dismutase - OxO Oxalate oxidase - PBS Phosphate-buffered saline     

Influence of Ca<Superscript>2+</Superscript> ions on metabolism of active oxygen species in wheat calli cocultured with the bunt pathogen <Emphasis Type="Italic">Tilletia caries</Emphasis>

The effect of Ca²⁺ on morphophysiological parameters of wheat calli (Triticum aestivum L.) infected by the bunt pathogen Tilletia caries, in particular on the level of active oxygen species, activity of oxalate oxidase, peroxidase, and catalase is investigated. The concentration of O²⁻, H₂O₂, and activity of oxidoreductases (oxalate oxidase, peroxidase, and catalase) depended on the content of Ca²⁺ in the culture medium of calli. The increase of the concentration of Ca²⁺ ions in the culture medium led to forming of calli with high structure, induction of activity of oxalate oxidase and of some isoperoxidase, and to accumulation of active oxygen species. These changes contributed to inhibition of development of the fungus. So this dependence confirm the role of calcium as the intermediant in biochemical reactions related to the formation of the protective response of plant cells to biotic stress.     

Effects of Liming on Potential Oxalate Secretion and Iron Chelation of Beech Ectomycorrhizal Root Tips

Liming is used to counteract forest decline induced by soil acidification. It consists of Ca and Mg input to forest soil and not only restores tree mineral nutrition but also modifies the availability of nutrients in soil. Ectomycorrhizal (ECM) fungi are involved in mineral nutrient uptake by trees and can recover them through dissolution of mineral surface. Oxalate and siderophore secretion are considered as the main agents of mineral weathering by ECMs. Here, we studied the effects of liming on the potential oxalate secretion and iron complexation by individual beech ECM root tips. Results show that freshly excised Lactarius subdulcis root tips from limed plots presented a high potential oxalate exudation of 177 μM tip⁻¹ h⁻¹. As this ECM species distribution is very dense, it is likely that, in the field, oxalate concentrations in the vicinity of its clusters could be very high. This points out that not only extraradical mycelium but also ECM root tips of certain species can contribute significantly to mineral weathering. Nonmetric multidimensional scaling (NMDS) separated potential oxalate production by ECM root tips in limed and untreated plots, and this activity was mainly driven by L. subdulcis ECMs, but NMDS on potential activity of iron mobilization by ECM root tips did not show a difference between limed and untreated plots. As the mean oxalate secretion did not significantly correlated with the mean iron mobilization by ECM morphotype, we conclude that iron complexation was due to either other organic acids or to siderophores.     

Germins and germin like proteins: an overview

Molecular investigations during wheat germination have revealed unique developmentally regulated proteins, designated as germins, which show remarkable resistance to broad specificity proteases and to dissociation in SDS. Germins in cereals have an oxalate oxidase activity, which generates H2O2 from the oxidative breakdown of oxalate thereby playing a significant role in plant development and defense. Germin like proteins (GLPs) exhibit sequence and structural similarity with the cereal germins but mostly lack oxalate oxidase activity. Germins and germin like proteins (GLPs) are a class of developmentally regulated glycoproteins characterized by a beta-barrel core structure, a signal peptide, and are associated with the cell wall. GLPs exhibit a broad range of diversity in their occurrence and activity in organisms ranging from myxomycetes, bryophytes, pteridophytes, gymnosperms and angiosperms. Germins and GLPs are thought to play a significant role during zygotic and somatic embryogenesis (wheat and Pinus, respectively), salt stress (barley and Mesembryanthemum crystallinum), pathogen elicitation (wheat and barley), and heavy metal stress, etc. Characterization and cloning of some of the genes encoding germins and GLPs has facilitated a better understanding of their regulation and raised their potential of biotechnological application.     

Characterisation of pea cytosolic glutathione reductase expressed in transgenic tobacco

 Expression in transgenic tobacco (Nicotiana tabacum L.) of a pea (Pisum sativum L.) GOR2 cDNA, encoding an isoform of glutathione reductase (GOR2), resulted in a 3- to 7-fold elevation of total foliar glutathione reductase (GR) activity. The enzyme encoded by GOR2 was confirmed to be extraplastidial in organelle fractionation studies and was considered most likely to be localised in the cytosol. A partial purification of GOR2 was achieved but a standard affinity chromatography step, using adenosine-2′,5′-diphosphate-Sepharose and often employed in the purification of GR from diverse sources, was unsuccessful with this isoform. Preparative isoelectric focussing was employed as part of the purification procedure of GOR2 and a complete separation from plastidial/mitochondrial glutathione reductase (GOR1) was achieved. The isoform GOR2 was shown to have a slower migration on non-denaturing polyacrylamide gels compared with GOR1 and properties typical of GR enzymes from plant sources. Received: 9 November 1999 / Accepted: 28 February 2000     

Transient expression of members of the germin-like gene family in epidermal cells of wheat confers disease resistance

The wheat genome encodes a family of germin-like proteins that differ with respect to regulation and tissue specificity of expression of the corresponding genes. While germin exhibits oxalate oxidase (E.C. 1.2.3.4.) activity, the germin-like proteins (GLPs) have no known enzymatic activity. A role of oxalate oxidase in plant defence has been proposed, based on the capacity of the enzyme to produce H2O2, a reactive oxygen species. The role in defence of germin and other members of the germin-like gene family was functionally assessed in a transient assay system based on particle bombardment of wheat leaves. Transient expression of the pathogen-induced germin gf-2.8 gene, but not of the constitutively expressed HvGLP1 gene, reduced the penetration efficiency of Blumeria (syn. Erysiphe) graminis f.sp. tritici, the causal agent of wheat powdery mildew, on transformed cells. Two engineered germin-gf-2.8 genes and the TaGLP2a gene, which all encoded proteins without oxalate oxidase activity, also reduced the penetration efficiency of the fungus, demonstrating that oxalate oxidase activity is not required for conferring enhanced resistance. Instead, activity tagging experiments showed that in cells transiently expressing the germin gf-2.8 gene, the transgene product became insolubilized at sites of attempted fungal penetration where localised production of H2O2 was observed. Thus, germin and GLPs may play a structural role in cell-wall re-enforcement during pathogen attack.     

New extracellular thermostable oxalate oxidase produced from endophytic Ochrobactrum intermedium CL6: Purification and biochemical characterization

Oxalate oxidase (EC 1.2.3.4) catalyzes the oxidative cleavage of oxalate to carbon dioxide with the reduction of molecular oxygen to hydrogen peroxide. Oxalate oxidase found its application in clinical assay for oxalate in blood and urine. This study describes the purification and biochemical characterization of an oxalate oxidase produced from an endophytic bacterium, Ochrobactrum intermedium CL6. The cell-free fermentation broth was subjected to two-step enzyme purification, which resulted in a 58.74-fold purification with 83% recovery. Specific activity of the final purified enzyme was 26.78 U?mg^?1 protein. The enzyme displayed an optimum pH and temperature of 3.8 and 80°C, respectively, and high stability at 4–80°C for 6?h. The enzymatic activity was not influenced by metal ions and chemical agents (K⁺, Na⁺, Zn²⁺, Fe³⁺, Mn²⁺, Mg²⁺, glucose, urea, lactate) commonly found in serum and urine, with Cu²⁺ being the exception. The enzyme appears to be a metalloprotein stimulated by Ca²⁺ and Fe²⁺. Its K_m and K_cat for oxalate were found to be 0.45?mM and 85?s^?1, respectively. This enzyme is the only known oxalate oxidase which did not show substrate inhibition up to a substrate concentration of 50?mM. Thermostability, kinetic properties, and the absence of substrate inhibition make this enzyme an ideal candidate for clinical applications.     

Identification and Characterization of a Multigene Family Encoding Germin-Like Proteins in Cultivated Peanut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.)

Germin-like proteins (GLPs) play diversified roles in plant development and defense response. Here, we identified 36 expressed sequence tags (ESTs) encoding GLPs from peanut (Arachis hypogaea L.). After assembly, these ESTs were integrated into eight unigenes named AhGLP1 to AhGLP8, of which, three (AhGLP1-3) were comprised 14, ten, and seven EST clones, respectively, whereas the remaining ones were associated with one single clone. The length of the deduced amino acid (AA) residues ranged from 208 to 223 AAs except for AhGLP6 and AhGLP8, which were incomplete at the carboxyl terminus. All of the AhGLPs contained a possible N-terminal signal peptide that was 17 to 24 residues in length excluding AhGLP7, where there is likely a non-cleavable amino terminus. Phylogenetic analysis showed that these AhGLPs were classified into three subfamilies. Southern blot analysis indicated that AhGLP1 and AhGLP2 likely have multiple copies in the peanut genome. The recombinant mature AhGLP1 and AhGLP2 proteins were successfully expressed in Escherichia coli. The purified AhGLP2 has superoxide dismutase (SOD) activity in enzymatic assay, but not oxalate oxidase activity. The SOD activity of AhGLP2 was stable up to 70°C and resistant to hydrogen peroxide, suggesting that AhGLP2 might be a manganese-containing SOD. Furthermore, AhGLP2 could confer E. coli resistance to oxidative damage caused by paraquat, suggesting that the AhGLP2 likely protects peanut plants from reactive oxygen metabolites. Thus, information provided in this study indicates the diverse nature of the peanut GLP family and suggests that some of AhGLPs might be involved in plant defense response.     

Proteolytic cleavage of plant proteins by peroxisomal endoproteases from senescent pea leaves

The degradation of peroxisomal and nonperoxisomal proteins by endoproteases of purified peroxisomes from senescent pea (Pisum sativum L.) leaves has been investigated. In our experimental conditions, most peroxisomal proteins were endoproteolytically degraded. This cleavage was prevented, to some extent, by incubation with 2 mM phenylmethylsulfonylfluoride, an inhibitor of serine proteinases. The peroxisomal enzymes glycolate oxidase (EC 1.1.3.1), catalase (EC 1.11.1.6) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) were susceptible to proteolytic degradation by peroxisomal endoproteases, whereas peroxisomal manganese superoxide dismutase (EC 1.15.1.1) was not. Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) from spinach and urease (EC 3.5.1.5) from jack bean were strongly degraded in the presence of peroxisomal matrices. These results indicate that proteases from plant peroxisomes might play an important role in the turnover of peroxisomal proteins during senescence, as well as in the turnover of proteins located in other cell compartments during advanced stages of senescence. On the other hand, our data show that peroxisomal endoproteases could potentially carry out the partial proteolysis which results in the irreversible conversion of xanthine dehydrogenase into the superoxide-generating xanthine oxidase (EC 1.1.3.22). This suggests a possible involvement of the peroxisomal endoproteases in a regulated modification of proteins. Received: 25 January 1999 / Accepted: 3 June 1999     

Stimulation of Root Formation onImpatiens balsamina L. cuttings by coumarin and the associated biochemical changes

Coumarin-caused stimulation of rooting of cuttings ofImpatiens balsamina L. is associated with an increase in the endogenous contents of carbohydrates, total phenols and proteins. Peroxidase, IAA oxidase and polyphenol oxidase activities also increased in coumarin treated cuttings at the time of cell division preceding primordia formation (6–12 h after treatment). Coumarin effect on rooting as well as on related biochemical changes resembles the effect of auxins in some ways.     

Farnesylation is involved in meristem organization in Arabidopsis

 Although studies in plant and animal cell culture systems indicate farnesylation is required for normal cell cycle progression, how this lipid modification of select proteins translates into whole-organism developmental decisions involving cell proliferation or differentiation is largely unknown. The era1 mutant of the higher plant Arabidopsis thaliana (L.) Heynh. offers a unique opportunity to understand the role farnesylation may play in regulating various processes during the development of a multicellular organism. Loss of farnesylation affects many aspects of Arabidopsis growth and development. In particular, apical and axillary meristem development is altered and these phenotypes are contingent on the growth conditions. Received: 25 October 1999 / Accepted: 22 December 1999     

A comparison of two-dimensional electrophoresis data with phenotypical traits in Arabidopsis leads to the identification of a mutant (cri1) that accumulates cytokinins

Total proteins extracted from developmental mutants of Arabidopsis thaliana (L.) Heyhn. and from wild-type plants cultivated in the presence of various hormones were analyzed by two-dimensional (2-D) gel electrophoresis. Computer analysis of 2-D gels followed by a statistical treatment of data allowed us to build a phenogram that describes the biochemical distances between the different genotypes. Analysis of the 2-D electrophoresis data allowed us to discriminate mutants in agreement with phenotypical and physiological traits. This biochemical analysis helped us to develop a working hypothesis which led us to show that one developmental mutant (cri1 ) overaccumulates cytokinins. Received: 5 August 1996 / Accepted: 11 December 1996     

Synthesis of oxalic Acid by enzymes from lettuce leaves

A rapid purification of lactate dehydrogenase and glycolate oxidase from lettuce (Lactuca sativa) leaves is described. The kinetics of both enzymes are reported in relation to their possible roles in the production of oxalate. Lettuce lactate dehydrogenase behaves like mammalian dehydrogenase, catalyzing the dismutation of glyoxylate to glycolate and oxalate. A model is proposed in which glycolate oxidase in the peroxisomes and lactate dehydrogenase in the cytosol are involved in the production of oxalate. The effect of pH on the balance between oxalate and glycolate produced from glyoxylate suggests that in leaves lactate dehydrogenase may function as part of an oxalate-based biochemical, pH-stat.     

Extracellular L-glutamate oxidase ofStreptomyces sp. Z-11-6: Obtainment and properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain,Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6–1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase fromStreptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1–0.5% promoted the secretion of the extracellular glutamate oxidase.     

Salicylic and Jasmonic acids in regulation of the proantioxidant state in wheat leaves infected by <Emphasis Type="Italic">Septoria nodorum</Emphasis> Berk

Influence of mediators of the signal systems of salicylic (SA) and jasmonic (JA) acids and their mixture on reactive oxygen species’ (ROS) (superoxide radical and O₂^·− H₂O₂) generation and activity of oxidoreductases (oxalate oxidase, peroxidase and catalase) in leaves of wheat Triticum aestivum L. infected by Septoria leaf blotch pathogen Septoria nodorum Berk has been studied. Presowing treatment of seeds by SA and JA decreased the development rate of fungus on wheat leaves. SA provided earlier inductive effect on production of O₂^·− and H₂O₂ compared with JA. The protective effect of the salicylic and jasmonic acids against Septoria leaf blotch pathogen was caused by activation of oxalate oxidase, induction of anion and cation peroxidases, and decrease of catalase activity. Ability of compounds to stimulate ROS in the plant tissues can be used as criteria for evaluation of immune-modulating activity of new substances for protection of the plants.     

Binding of Selected Extracellular Matrix Proteins to Enterococci and Streptococcus bovis of Animal Origin

Thirty-three enterococcal strains and 10 Streptococcus bovis strains were investigated for their protein-binding cell surface components. Seven extracellular matrix (ECM) proteins were immobilized on Difco latex beads to detect these components on the surface of all enterococcal strains and eight non-autoaggregating S. bovis strains by a particle agglutination assay (PAA). Twenty-three selected strains were also examined in microtiter plate assays. According to the absorbance readings (A_570nm), 11 strains were classified as nonadherent (A_570nm < 0.1), 10 strains as weakly adherent (0.1 < A_570nm > 0.3), and 2 strains as strongly adherent (A_570nm > 0.3) in these assays. A direct correlation was found between the values obtained in PAA and A_570nm readings of microtiter plate assays. Binding of ¹²⁵I-labeled bovine lactoferrin to enterococci and streptococci was in the range of 6%–30% and of ¹²⁵I-labeled human vitronectin in the range of 9%–33% to streptococci. The binding of ¹²⁵I-labeled ECM proteins to selected strains was much more effectively inhibited by sulfated carbohydrates than by non-sulfated hyaluronic acid, indicating the importance of the sulfate groups of these inhibitors. An inhibition effect of heparin on bLf binding to four selected strains was higher in comparison with fucoidan in the microtiter plates. Thirty-five out of 44 strains had agglutinated rabbit erythrocytes. However, these strains showed no ability to agglutinate bovine or sheep erythrocytes. Received: 28 April 1999 / Accepted: 26 July 1999     

Protein- and pH-dependent binding of nascent pectin and glucuronoarabinoxylan to xyloglucan in pea

 Nascent pectin and glucuronoarabinoxylan, synthesised in vitro by membrane-bound enzymes from etiolated pea (Pisum sativum L.) epicotyls, were found to bind to pea xyloglucan in a pH-dependent manner. The binding was maximum at low pH (3–4), and decreased to almost zero at pH 6. The binding was probably non-covalent and reached saturation within 5 min. Removal of the fucose residues of xyloglucan decreased the degree of binding. Removal by protease of the proteins attached to nascent pectin and glucuronoarabinoxylan greatly reduced the maximum binding and abolished the pH-dependence. The observed binding may be of considerable significance in the process of cell-wall assembly and in the control of cell extension. Received: 4 November 1999 / 22 December 1999     

Microscale spatial distribution patterns of red oak (Quercus rubra L.) ectomycorrhizae

The microscale spatial distribution patterns of ectomycorrhizal (ECM) morphotypes of red oak (Quercus rubra L.) were analyzed over a 600 × 6 × 3 cm (length x width x depth) soil monolith. For this purpose, the soil monolith was divided into 2 × 2 × 1 cm cuboids. Each cuboid was assigned to an organic sublayer, namely the F- or H-layer. A new classification method was used to combine morphotypes with similar distribution patterns into five different groups. For identification of the ECM fungi, internal transcribed spacer (ITS) regions from nuclear rDNA were sequenced and compared with sequences from the international GenBank. Twenty-eight ECM types were identified in the soil monolith. Using this new classification method, it was found that the majority of morphotypes (16) appeared in a scattered horizontal distribution and that only 5 morphotypes formed clusters. In addition, it was found that many morphotypes had a clear preference for a specific organic sublayer: 14 morphotypes preferred the F-layer, 5 the H-layer, and only 4 morphotypes showed no preference for a given layer. Analysis of the distribution patterns showed that ECM fungi either exclude each other or occur together. The most frequent morphotypes, Cenococcum geophilum and Tomentella spec. 01, were associated with the whole ECM community. In contrast, the frequent morphotype Tomentella terrestris showed the lowest degree of association among the identified morphotypes.     

Germin and Germin-like Proteins: Evolution,Structure, and Function

Germin and germin-like proteins (GLPs) are encoded by a family of genes found in all plants. They are part of the cupin superfamily of biochemically diverse proteins, a superfamily that has a conserved tertiary structure, though with limited similarity in primary sequence. The subgroups of GLPs have different enzyme functions that include the two hydrogen peroxide–generating enzymes, oxalate oxidase (OxO) and superoxide dismutase. This review summarizes the sequence and structural details of GLPs and also discusses their evolutionary progression, particularly their amplification in gene number during the evolution of the land plants. In terms of function, the GLPs are known to be differentially expressed during specific periods of plant growth and development, a pattern of evolutionary subfunctionalization. They are also implicated in the response of plants to biotic (viruses, bacteria, mycorrhizae, fungi, insects, nematodes, and parasitic plants) and abiotic (salt, heat/cold, drought, nutrient, and metal) stress. Most detailed data come from studies of fungal pathogenesis in cereals. This involvement with the protection of plants from environmental stress of various types has led to numerous plant breeding studies that have found links between GLPs and QTLs for disease and stress resistance. In addition the OxO enzyme has considerable commercial significance, based principally on its use in the medical diagnosis of oxalate concentration in plasma and urine. Finally, this review provides information on the nutritional importance of these proteins in the human diet, as several members are known to be allergenic, a feature related to their thermal stability and evolutionary connection to the seed storage proteins, also members of the cupin superfamily.