Pathogenesis-related acidic beta-1,3-glucanase genes of tobacco are regulated by both stress and developmental signals

Three pathogenesis-related (PR) proteins of tobacco are acidic isoforms of beta-1,3-glucanase (PR-2a, -2b, -2c). We have cloned and sequenced a partial cDNA clone (lambda FJ1) corresponding to one of the PR-2 beta-1,3-glucanases. A small gene family encodes the PR-2 proteins in tobacco, and similar genes are present in a number of plant species. We analyzed the stress and developmental regulation of the tobacco PR-2 beta-1,3-glucanases by using northern and western analyses and a new technique to assay enzymatic activity. Stress caused by both thiamine and tobacco mosaic virus (TMV) infection resulted in a dramatic increase in the levels of PR-2 mRNA, protein, and enzyme activities. The increased PR-2 gene expression in upper uninoculated leaves of plants infected with TMV also suggests a role in systemic acquired resistance. During floral development, a number of beta-1,3-glucanase activities were observed in all flower tissues. However, PR-2 polypeptides were observed only in sepal tissue. In contrast, an mRNA that hybridized to the PR-2 cDNA was present in stigma/style tissue and the sepals. Primer extension analysis confirmed the identity of the PR-2 mRNA in sepals, but indicated that the beta-1,3-glucanase gene expressed in the stigma/style of flowers was distinct from the PR-2 genes. The induction of PR-2 protein synthesis by both stress and developmental signals was accompanied by a corresponding increase in the steady-state levels of PR-2 mRNA, suggesting that PR-2 gene expression is regulated, in part, at the level of mRNA accumulation.     

A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis

Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be determined by beta-1,3-glucan synthase versus glucanase activities. A proteomic analysis of an Arabidopsis thaliana plasmodesmata (Pd)-rich fraction identified a beta-1,3-glucanase as present in this fraction. The protein encoded by the putative plasmodesmal associated protein (ppap) gene, termed AtBG_ppap, had previously been found to be a post-translationally modified glycosylphosphatidylinositol (GPI) lipid-anchored protein. When fused to green fluorescent protein (GFP) and expressed in tobacco (Nicotiana tabacum) or Nicotiana benthamiana epidermal cells, this protein displays fluorescence patterns in the endoplasmic reticulum (ER) membrane system, along the cell periphery and in a punctate pattern that co-localizes with aniline blue-stained callose present around the Pd. Plasma membrane localization was verified by co-localization of AtBG_ppap:GFP together with a plasma membrane marker N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) in plasmolysed cells. In Arabidopsis T-DNA insertion mutants that do not transcribe AtBG_ppap, functional studies showed that GFP cell-to-cell movement between epidermal cells is reduced, and the conductivity coefficient of Pd is lower. Measurements of callose levels around Pd after wounding revealed that callose accumulation in the mutant plants was higher. Taken together, we suggest that AtBG_ppap is a Pd-associated membrane protein involved in plasmodesmal callose degradation, and functions in the gating of Pd.     

Cloning and expression analysis of two beta-1,3-glucanase genes from strawberry

We isolated from strawberry (Fragariae x ananassa Duch) a genomic clone of a beta-1,3-glucanase gene, designated as FaBG2-2. In addition, a related cDNA clone, designated as FaBG2-3, was also isolated. FaBG2-2 and FaBG2-3 are similar in their coding regions, except that FaBG2-2 does not appear to contain a signal peptide coding sequence. The 5' and 3' flanking regions of FaBG2-2 and FaBG2-3 are differentt. Using real-time PCR, the expression patterns of FaBG2-3 and a previously isolated beta-1,3-glucanase gene, FaBG2-1, in strawberry plants infected with Colletotrichum fragariae or Colletotrichum acutatum were analyzed at different time points post-infection. The results showed that expressions of both genes in the leaves of infected plants were induced by the two fungi, but the level of induction was several fold greater with C. fragariae. Comparison of the expression levels of the two genes revealed that the level of FaBG2-3 expression was several hundred to over a thousand fold higher than that of FaBG2-1. Furthermore, the expression levels of the two genes in the leaf, fruit, crown and root of uninfected strawberry plants were analyzed.     

Antisense-transformation reveals novel roles for class I beta-1,3-glucanase in tobacco seed after-ripening and photodormancy

Little is known about the molecular basis for seed dormancy, after-ripening, and radicle emergence through the covering layers during germination. In tobacco, endosperm rupture occurs after testa rupture and is the limiting step in seed germination. Class I beta-1,3-glucanase (betaGLU I), which is induced in the micropylar endosperm just prior to its penetration by the radicle, is believed to help weaken the endosperm wall. Evidence is presented here for a second site of betaGLU I action during after-ripening. Tobacco plants were transformed with antisense betaGLU I constructs with promoters thought to direct endosperm-specific expression. Unexpectedly, these transformants were unaffected in endosperm rupture and did not exhibit reduced betaGLU I expression during germination. Nevertheless, antisense betaGLU I transformation delayed the onset of testa rupture in light-imbibed, after-ripened seeds and inhibited the after-ripening-mediated release of photodormancy. It is proposed that betaGLU I expression in the dry seed contributes to the after-ripening-mediated release of seed dormancy.     

Amperometric determination of laminarin using immobilized beta-1,3-glucanase

A novel sensor system equipped with a reactor packed with beads containing immobilized beta-1,3-glucanase and glucose oxidase was developed for the amperometric determination of laminarin concentration. The proposed sensor system consisted of a reactor, an oxygen electrode, a flow cell, a pump, a buffer tank, and a recorder. The measurement was performed with a flow injection system. The optimum conditions for the sensor system were as follows: transfer solution, pH 7.0; 0.1 M phosphate buffer solution; flow rate, 0.15 ml/min; and sample volume, 50 microl. The response was correlated to the laminarin concentration. The calibration curve was obtained between 50 and 0.5 mg/ml laminarin (R2 = 0.994). The detection limit was 50 microg/ml laminarin (the ratio of signal/noise = 3). The relative standard deviations were 2.0% (n = 15) and 2.5% (n = 15) for 0.4 and 1.0 mg/ml laminarin solutions, respectively. One assay was completed within 5 min. Results suggest that the sensor can be used not only for the analysis of seaweed and health-enhancing foods but also for monitoring the initial pollution of the marine environment.     

Regulation of beta-1,3-glucanase synthesis in Penicillium italicum.

The filamentous fungus Penicillium italicum produced a certain level of beta-1,3-glucanase during active growth in a glucose-supplemented medium; however, at a low glucose concentration (2 to 10 mM), derepression took place and the specific activity of the enzyme increased significantly. Derepressed cells (incubated in a glucose-limited medium) accumulated a capacity for the synthesis of beta-1,3-glucanase, which led to a subsequent increase in the specific activity even when the cells were transferred to a medium with an excess of glucose (180 mM). Two protein synthesis inhibitors, cycloheximide and trichodermin, immediately stopped the increase in specific activity when added to derepressed cells. On the other hand, 8-hydroxyquinoline, an RNA a synthesis inhibitor, acted differently, since it permitted the specific activity to increase for some time after being added to depressed cells. Moreover, the concentration of glucose did not affect the 8-hydroxyquinoline-insensitive synthesis of beta-1,3-glucanase. It is concluded that the glucose repression effect on beta-1,3-glucanase production must be exerted at a pretranslational level that could be either mRNA synthesis or some stage of the process involved in its maturation or stabilization.     

Development of beta-1,3-glucanase activity in germinated tomato seeds

Laminarin-hydrolysing activity developed in the endosperm of tomato (Lycopersicon esculentum) seeds following germination. The enzyme was basic (pI>10) and the apparent molecular mass was estimated to be 35 kDa by SDS-PAGE. It was specific for linear beta-1,3-glucan substrates. Laminarin was hydrolysed by the enzyme to yield a mixture of oligoglucosides, indicating that the enzyme had an endo-action pattern. Thus, the enzyme was identified as beta-1,3- endoglucanase (EC 3.2.1.39). The activity of the enzyme developed in the endosperm after radicle protrusion (germination) had occurred and the enzyme activity was localized exclusively in the micropylar region of the endosperm where the radicle had penetrated. When the lateral endosperm region, where no induction of the enzyme occurred, was wounded (cut or punctured), there was a marked enhancement of beta-1,3-glucanase activity. Thus the post-germinative beta-1, 3-glucanase activity in the micropylar endosperm portion might be brought about by wounding resulting from endosperm rupture by radicle penetration.     

Sense transformation reveals a novel role for class I beta-1, 3-glucanase in tobacco seed germination

'Coat-enhanced' seed dormancy of many dicotyledonous species, including tobacco, is released during after-ripening. Rupture of the endosperm, which is the limiting step in tobacco seed germination, is preceded by induction of class I beta-1,3-glucanase (betaGLU I) in the micropylar endosperm where the radicle will penetrate. Treating after-ripened tobacco seeds with abscisic acid (ABA) delays endosperm rupture and inhibits betaGLU I induction. Sense transformation with a chimeric ABA-inducible betaGLU I transgene resulted in over-expression of betaGLU I in seeds and promoted endosperm rupture of mature seeds and of ABA-treated after-ripened seeds. Taken together, these results provide direct evidence that betaGLU I contributes to endosperm rupture. Over-expression of betaGLU I during germination also replaced the effects of after-ripening on endosperm rupture. This suggests that regulation of betaGLU I by ABA signalling pathways might have a key role in after-ripening.     

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.     

Three N-terminal domains of beta-1,3-glucanase A1 are involved in binding to insoluble beta-1,3-glucan.

Limited proteolysis of beta-1,3-glucanase A1 by three different proteases, trypsin, chymotrypsin, and papain, gave three major active fragments. The sizes of the three major fragments generated by each protease treatment were identical to those of beta-1,3-glucanase A2, A3, and A4 detected in both the culture supernatant of Bacillus circulans WL-12 and the periplasmic space of Escherichia coli carrying a cloned glcA gene. These results indicate a four-domain structure for the enzyme. At the N terminus of the glucanase, duplicated segments of approximately 100 amino acids were observed. N-terminal amino acid sequence analysis revealed that the active fragments with sizes corresponding to those of A2 and A3 lack the first segment (domain) and both duplicated segments (domains), respectively. The fragment corresponding to A4 lacks both duplicated segments and the following ca. 120-amino-acid region. By losing the first, second, and third (corresponding to the segment of 120 amino acids) domains, beta-1,3-glucanase progressively lost the ability to bind to pachyman, beta-1,3-glucan. An active fragment which did not have the three N-terminal domains did not show significant binding to pachyman. Thus, all three N-terminal domains contribute to binding to beta-1,3-glucan, and the presence of three domains confers the highest binding activity on the glucanase. The loss of these binding domains remarkably decreased pachyman-hydrolyzing activity, indicating that the binding activity is essential for the efficient hydrolysis of insoluble beta-1,3-glucan.     

Chitinase, beta-1,3-glucanase, osmotin, and extensin are expressed in tobacco explants during flower formation.

Sequence analysis of five gene families that were isolated from tobacco thin cell layer explants initiating floral development [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35] showed that two encode the pathogenesis-related proteins basic chitinase and basic beta-1,3-glucanase, while a third encodes the cell wall protein extensin, which also accumulates during pathogen attack. Another sequence family encodes the water stress-induced protein osmotin [Singh et al. (1989). Plant Physiol. 90, 1096-1101]. We found that osmotin was also induced by viral infection and wounding and, hence, could be considered a pathogenesis-related protein. These genes, which were highly expressed in explants during de novo flower formation but not in explants forming vegetative shoots [Meeks-Wagner et al. (1989). Plant Cell 1, 25-35], were also regulated developmentally in day-neutral and photoresponsive tobacco plants with high expression levels in the roots and moderate- to low-level expression in other plant organs including flowers. An unidentified gene family, FB7-4, had its highest level of expression in the basal internodes. Our findings indicate that these genes, some of which are conventionally considered to encode pathogen-related proteins, also have a complex association with normal developmental processes, including the floral response, in healthy plants.     

Control of Extracellular beta-1,3-glucanase activity in a basidiomycete species.

The basidiomycete QM 806 excreted large amounts of beta-1,3-glucanase into the culture medium. Synthesis and excretion of the enzyme were triggered by a critically low concentration of carbon source. The extracellular beta-1,3-glucanase exhibited a remarkable stability. Addition of glucose or other carbon sources to a culture after consumption of the initial carbon source led to an inactivation of the extracellular beta-1,3-glucanase by an inactivating system, which could be separated from the cells. The inactivation of beta-1,3-glucanse was prevented by cycloheximide. This indicates the necessity of active protein synthesis for the inactivation process but does not prove that the inactivating system itself is a protein. Marked changes in the electrophoretic mobility and immunological properties of beta-1,3-glucanase indicate rather profound alterations of the enzyme protein in the course of inactivation.     

Streptomyces matensis laminaripentaose hydrolase is an 'inverting' beta-1,3-glucanase.

The laminaripentaose-producing beta-1,3-glucanase of Streptomyces matensis is a member of the glycoside hydrolase family GH-64. We have constructed and purified a recombinant hexahistidine-tagged form of the enzyme for characterisation. The enzyme, which exists as a monomer in solution, hydrolyses beta-1,3-glucan by a mechanism leading to overall inversion of the anomeric configuration. This is the first determination of the mechanism prevailing in glycoside hydrolase family GH-64 and this is the first characterisation of an 'inverting' beta-1,3-glucanase.     

Characterization of a 29-kDa beta-1,3-glucanase from Trichoderma harzianum

A beta-1,3-glucanase, from culture filtrates of Trichoderma harzianum, was purified in sequential steps by gel filtration, hydrophobic interaction and ion exchange chromatography. A typical procedure provided 69-fold purification with 0.32% yield. The molecular mass of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on a 10% slab gel. The K(M) and V(max) values for beta-1,3-glucanase, using laminarin as substrate, were 1. 72 mg ml(-1) and 3.10 U ml(-1), respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 50 degrees C. The enzyme was strongly inhibited by HgCl(2) and SDS. These results suggest that each beta-1,3-glucanase produced by T. harzianum is different and is probably encoded by different genes.     

Chemical modification of lysine residues of beta-1,3-glucanase from Spisula sachalinensis

The effects of chemical modification of the amino groups of lysine residues on the activity of beta-1.3-glucanase from Spisula sachalinensis were studied. Modification of two lysine residues per molecule did not affect either the enzyme activity with respect to laminarine, nor the Km value. The modified beta-1.3-glucanase retains the ability to catalyze the transglycosylation and cleaves the high molecular weight CM-pachyman at the same rate as does the native enzyme. No significant changes in the enzyme thermal stability were observed. Thus, the modified enzyme groups cannot be involved in the enzyme active center and are exposed on the surface of the protein globule. The chemical modification was shown to have no effect on the enzyme kinetics, which is essential for its immobilization.     

Seed after-ripening and over-expression of class I beta-1,3-glucanase confer maternal effects on tobacco testa rupture and dormancy release

'Coat-imposed' seed dormancy of many non-endospermic and endospermic species is released during after-ripening. After-ripening-mediated promotion of tobacco ( Nicotiana tabacum L.) seed germination is mainly due to a promotion of testa rupture and a similar promotion of subsequent endosperm rupture. Treatment of after-ripened or freshly harvested mature seeds with abscisic acid (ABA) delays endosperm rupture and inhibits the induction of class I beta-1,3-glucanase (betaGlu I) in the micropylar endosperm, but does not affect the kinetics of testa rupture. After-ripening-mediated release of photodormancy is correlated with a decreased gibberellin (GA) requirement for testa rupture during dark-imbibition. Reciprocal crosses between wild-type tobacco and sense-betaGlu I transformant lines showed that betaGlu I over-expression in the seed covering layers can replace the promoting effect of after-ripening on testa rupture in light, but only if the mother plant is a sense-betaGlu I line. This maternal effect supports the model of two sites for betaGlu I action: (i) betaGlu I contribution to the after-ripening-mediated release of dormancy in the dry seed state, which is manifested in the promotion and ABA-insensitivity of testa rupture during imbibition. (ii) ABA-sensitive expression of betaGlu I in the micropylar endosperm, which contributes to endosperm rupture. The importance of GA-signaling and testa characteristics appear to be a common feature during the after-ripening-mediated release of coat-imposed dormancy in endospermic and non-endospermic seeds.