A rice ��-1,3-glucanase gene Osg1 is required for callose degradation in pollen development

Plant β-1,3-glucanases are involved in plant defense and development. In rice (Oryza sativa), 14 genes encoding putative β-1,3-glucanases have been isolated and sequenced. However, only limited information is available on the function of these β-1,3-glucanase genes. In this study, we report a detailed functional characterization of one of these genes, Osg1. Osg1 encodes a glucanase carrying no C-terminal extension. Osg1 was found to be expressed throughout the plant and highly expressed in florets, leaf sheaths, and leaf blades. Investigations using real-time PCR, immunocytochemical analysis, and a GUS-reporter gene driven by the Osg1 promoter indicated that Osg1 was mainly expressed at the late meiosis, early microspore, and middle microspore stages in the florets. To elucidate the role of Osg1, we suppressed expression of the Osg1 gene by RNA interference in transgenic rice. The silencing of Osg1 resulted in male sterility. The pollen mother cells appeared to be normal in Osg1-RI plants, but callose degradation was disrupted around the microspores in the anther locules of the Osg1-RI plants at the early microspore stage. Consequently, the release of the young microspores into the anther locules was delayed, and the microspores began to degenerate later. These results provide evidence that Osg1 is essential for timely callose degradation in the process of tetrad dissolution.     

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.     

Proteomic analysis of β-1,3-glucanase in grape berry tissues

Grape berries are considered recalcitrant materials in proteomic analysis, because berry tissues contain large amounts of secondary metabolites, especially phenolic compounds, which severely interfere with protein extraction and electrophoresis separation. We report hereby a PVPP/TCA-based protein extraction protocol for grape berries. Phenolic compounds in berry extracts were removed with repeated PVPP cleanups, and proteins were recovered with TCA precipitation. Protein resolution in 2-D gels was gradually improved with the increase of PVPP cleanup steps. By the protocol, about 760 protein spots of berry tissues were clearly resolved in 2-D gels with CBB staining. This protocol was also used to analyze β-1,3-glucanase (EC 3.2.1.39) in berry tissues. An anti-synthetic peptide antibody was prepared against 15 amino acid sequence residing on the surface of β-1,3-glucanase molecule. It detected two major spots in 2-D blots of berry extracts. The spots were identified by MALDI-TOF analysis as β-1,3-glucanase. The present study validates that β-1,3-glucanase is present in higher abundance in berry skins than in pulps, and in red berries than in white berries. Therefore, β-1,3-glucanase displays a tissue-specific expression. The preferential accumulation of β-1,3-glucanase in skins may be relevant to berry ripening.     

Purification and Some Properties of Urethane Hydrolase II from Lactobacillus casei ω ATCC 7469

The β-1,3-glucanase (1,3-β-d-glucan glucanohydrolase, EC 3.2.1.6) gene from Flavobacterium dormitator var. glucanolyticae was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKUβG1 (8.2 kb), showed a high β-1,3-glucanase activity and a lytic activity on viable yeast cells. These activities were found in the peripiasmic space of E. coli clone cells. Southern hybridization analysis showed that the cloned gene was derived from F. dormitator chromosomal DNA. The gene products were purified from the periplasmic fraction of E. coli by ammonium sulfate fractionation and ion-exchange chromatography. The purified enzymes were demonstrated to be identical with a lytic endo-β-1,3-glucanase II and a nonlytic endo-β-1,3-glucanase I from F. dormitator from their enzymological and immunological properties. In the E. coli cells, endo-β-1,3-glucanase I was also formed by a proteolytic digestion of endo-β-1,3-glucanase II during the cultivation as in F. dormitator. Thus, the only endo-β-1,3-glucanase II was coded for in the cloned gene.     

Tapetum-Specific Expression of the Gene for an Endo-{beta}-1,3-glucanase Causes Male Sterility in Transgenic Tobacco

A cDNA for a pathogenesis-related endo-ß-1,3-glucanaseisolated from soybean, was fused to an anther tapetum-specificpromoter (Osg6B promoter) isolated from rice and the resultingchimeric gene was introduced into tobacco. The Osg6B promoterbecame active in the anther tapetum during formation of tetradsand the tapetal glucanase activity in the transgenic plantscaused in a significant reduction in the number of fertile pollengrains. Most of the pollen grains were aberrant in shape, lackedgerminal apertures and aggregate of the pollen grains. Granulesof ß-1,3-glucan, which have not previously been reported,were often observed to adhere to the surface of the pollen grains.Further observations revealed that the callose wall was almostabsent in the pollen tetrads of transgenic plants. In wild-typeplants, by contrast, the tetrads were surrounded by callosethat was degraded soon after the tetrad stage to release freemicrospores. Thus, the introduced gene for endo-ß-1,3-endoglucanaseunder the control of the Osg6B promoter caused digestion ofthe callose wall at the beginning of the tetrad stage, a timethat was just a little earlier than the time at which endogenousglucanase activity normal appears. These results demonstratethat premature dissolution of the callose wall in pollen tetradscauses male sterility and suggest that the time at which tapetallyproduced glucanase is activate is critical for the normal developmentof microspores. (Received September 29, 1994; Accepted January 30, 1995)     

生防木霉菌β-1,3-葡聚糖酶活性研究

通过采取还原糖法对生防木霉菌真菌2号和真菌4号菌株在不同发酵时间产β-1,3-葡聚糖酶活性进行测定,对其产β-1,3-葡聚糖酶特性进行初步研究。结果表明,同一菌株在不同发酵时间β-1,3-葡聚糖酶活性大小变化的趋势大致相同,并均在培养72 h时β-1,3-葡聚糖酶活性达到最大,在相同发酵时间,真菌2号菌株比真菌4号菌株的β-1,3-葡聚糖酶活性要高。     

Functional analysis of a beta-1,3-glucanase gene (Tag1) with anther-specific RNA and protein accumulation using antisense RNA inhibition

A critical stage in pollen development is the dissolution of tetrads into free microspores. Tetrads are surrounded by a wall composed primarily of beta-1,3-glucan. At the completion of meiosis, tetrads are released into the anther locule after hydrolysis of the callose by a beta-1,3-glucanase complex. The cDNA corresponding to a beta-1,3-glucanase cloned from tobacco (Tag 1) represents a gene that is highly similar to other beta-1,3-glucanases and is expressed exclusively in anthers from the tetrad to free microspore stage of pollen development. Tag 1 protein was overexpressed in E. coli, accumulating in insoluble inclusion bodies. Polyclonal antibodies against Tag 1 recombinant protein identify a single 33 kD protein accumulating only in anthers at tetrad and free microspore stages where beta-1,3-glucanase activity is present. Transgenic plants expressing Tag 1 antisense RNA were produced. Although Tag 1 RNA and protein levels were greatly reduced, tetrad dissolution and pollen development were normal. These data indicate that under the conditions these tobacco plants were grown, wild type levels of Tag 1 protein are not necessary for male fertility.     

Functional Implications of the Subcellular Localization of Ethylene-Induced Chitinase and [beta]-1,3-Glucanase in Bean Leaves

Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and [beta]-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and [beta]-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of [beta]-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. Both kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and [beta]-1,3-glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and [beta]-1,3-glucanase in isolated vacuoles, and demonstrated that only [beta]-1,3-glucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and [beta]-1,3-glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized [beta]-1,3-glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens.     

Inhibitory effect of 2-Deoxyglucose on cell wall alpha-1,3-glucan synthesis and cleistothecium development in Aspergillus nidulans

2-Deoxy-d-glucose (2-DG) (0.1% w/v) added at inoculation time to cultures of Aspergillus prevents α-1,3-glucan, α-1,3-glucanase, and cleistothecium formation. 2-DG given after α-1,3-glucan synthesis, inhibits α-1,3-glucanase and glucan breakdown partially, and cleistothecium formation completely, perhaps because 2-DG, a toxic analog, is the only sugar available to the organism. When 2-DG is provided after about half the α-1,3-glucan is already made, about half the amount of α-1,3-glucanase is synthesized. Cleistothecia do develop, probably because at the time of addition of 2-DG glucose is still available in the medium. Experiments designed to test whether 2-DG acts primarily on α-1,3-glucan, α-1,3-glucanase, or both show that extra glucose appears mainly in an alkali-insoluble fraction and only little is synthesized into α-1,3-glucan. This is the reverse of what happens in the absence of 2-DG. These results indicate that both α-1,3-glucan and α-1,3-glucanase are indispensable for fructification in Aspergillus. Moreover, 2-DG primarily influences the synthesis of α-1,3-glucan, and the inhibition of α-1,3-glucanase is either a secondary effect or a consequence of disturbing the cell's metabolism.     

Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family

The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).     

Production and catabolite repression of Penicillium italicum beta-glucanases.

The filamentous fungus Penicillium italicum, grown in a defined liquid medium, produced beta-1,3-glucanase, which remained essentially bound to the cells, and beta-1,6-glucanase, an essentially extracellular enzyme. When glucose was depleted from the medium, when a limited concentration of glucose (0.2%) was maintained, or when the carbon source was galactose (3%) or lactose (3%), a significant increase in the specific activity of beta-1,3-glucanase, in cell extracts, took place. This was paralleled by a very slow rate of growth, and under glucose limitation, the appearance of beta-1,3-glucanase in the medium was also observed. On the other hand, when an excess of glucose, fructose, or sucrose was present, the specific activity remained constant and active growth was promoted. Laminarin, cellobiose, gentiobiose, and isolated Penicillium italicum walls were not capable of significantly inducing beta-1,3-glucanase synthesis to a level beyond that attained by glucose limitation. A similar behavior was observed for beta-1,6-glucanase. beta-1,3-Glucanase and beta-1,6-glucanase are therefore constitutive enzymes subjected to catabolite repression. The results are discussed in the context of the possible functions that have been suggested for glucanases and related enzymes.     

Cloning and Expression of an α-1,3-Glucanase Gene from Bacillus circulans KA-304: The Enzyme Participates in Protoplast Formation of Schizophyllum commune

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of α-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity.

The gene of α-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of α-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of α-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (α-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases.

The recombinant α-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant α-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-β-D-thiogalactopyranoside. The recombinant α-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.     

Induction of β-1,3-glucanase in seeds of maize defective-kernel mutant (827Kpro1)

β-1,3-glucanases are found in organisms as diverse as plants, animals, bacteria and fungi. In plants, such enzymes are not only associated with defense mechanisms against pathogens, but also play critical roles in physiological and developmental processes. Here we identified a new β-1,3-glucanase in maize seeds, and named it ZmGlucA. Sequence analysis revealed that ZmGlucA belongs to the class A of β-1,3-glucanase, a class related to defense and physiological processes in plants. mRNA and protein assays showed that zmGlucA is expressed exclusively in seeds, and it is differentially regulated during seed development. Additionally, zmGlucA expression is strongly induced in seeds of the mutant dek 827Kpro1, which is defective for embryo and endosperm development. Our data support the idea that ZmGlucA protein is relevant to seed development.     

Hormonal regulation of beta1,3-glucanase messenger RNA levels in cultured tobacco tissues

We describe the isolation of a cDNA clone of β1,3-glucanase mRNA from Nicotiana tabacum L. cv. `Havana 425' and its use to measure the kinetics of mRNA accumulation in cultured tobacco tissues treated with the plant hormones auxin and cytokinin. Northern blot analysis showed that the tissues contain a single ˜1.6 kb-sized β1,3-glucanase mRNA. The levels of β1,3-glucanase and β1,3-glucanase mRNA increase by up to seven- and 20-fold, respectively, over a 7-day period in tissues subcultured on hormone-free medium and medium containing auxin or cytokinin added separately. Over the same interval of time, the content of both the enzyme and its mRNA remains at a constant low level in tissues subcultured on medium containing both auxin and cytokinin. The results show that auxin and cytokinin block β1,3-glucanase production at the level of the mRNA.     

Characterization of a chitinase and an endo-beta-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Of 24 Trichoderma isolates, T harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and beta-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa beta-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (beta-1,3-glucanase). Km and Kcat were 3.8 g l(-1) and 0.71 s(-1) for the chitinase (chitin) and 1.1 g(-1) and 52 s(-1) for the beta-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the beta-1,3-glucanase was specific for beta-1,3-linkages in polysaccharides. Both enzymes were stable at 30 degrees C, while at 60 degrees C the chitinase and the beta-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 microg/ml.     

Induction of beta-1,3-glucanase in barley in response to infection by fungal pathogens.

The sequence of a partial cDNA clone corresponding to an mRNA induced in leaves of barley (Hordeum vulgare) by infection with fungal pathogens matched almost perfectly with that of a cDNA clone coding for beta-1,-3-glucanase isolated from the scutellum of barley. Western blot analysis of intercellular proteins from near-isogenic barley lines inoculated with the powdery mildew fungus (Erysiphe graminis f. sp. hordei) showed a strong induction of glucanase in all inoculated lines but was most pronounced in two resistant lines. These data were confirmed by beta-1,3-glucanase assays. The barley cDNA was used as a hybridization probe to detect mRNAs in barley, wheat (Triticum aestivum), rice (oryza sativus), and sorghum (Sorghum bicolor), which are induced by infection with the necrotrophic pathogen Bipolaris sorokiniana. These results demonstrate that activation of beta-1,3-glucanase genes may be a general response of cereals to infection by fungal pathogens.