Co-ordinated regulation of chitinase and β-1,3-glucanase in bean leaves

Ethylene induced chitinase (EC 3.2.1.14) and -1,3-glucanase (EC 3.2.1.29) to a similar extent in primary leaves of bean seedlings (Phaseolus vulgaris cv. Saxa). Both enzymes were purified from ethylene-treated leaves, and monospecific antibodies were raised aginst them. Ethylene treatments strongly increased the amount of immunore-active chitinase and -1,3-glucanase. Ethylene enhanced synthesis of chitinase in vivo, as tested by immunoprecipitation after pulse-labelling with [³⁵S]methionine. RNA was isolated from bean leaves and translated in a rabbit reticulocyte lysate system in vitro. The chitinase and the -1,3-glucanase antiserum each precipitated a single polypeptide from the translation products. The precipitated polypeptides were 1500 and 4000 daltons larger, respectively, than native chitinase and native -1,3-glucanase, indicating that the two enzymes were synthesized as precursors in vitro. The translatable mRNAs for both enzymes increased at least tenfold within 2 h in response to a treatment with ethylene. When ethylene was withdrawn after 8 h of incubation, the translatable mRNAs for both enzymes decreased somewhat more slowly, reaching the basal level about 25 h later. In all cases, there was a close correlation between the levels of translatable mRNA for chitinase and -1,3-glucanase. A putative -1,3-glucanase cDNA clone, pCH16, was isolated by hybrid-selected translation. The amount of -1,3-glucanase mRNA, as measured by RNA blot analysis using pCH16 as a probe, increased rapidly in response to ethylene and decreased again after withdrawal of ethylene, indicating that the amount of hybridizable RNA and of translatable mRNA for -1,3-glucanase were correlated. In conclusion, the results indicate that chitinase and -1,3-glucanase are regulated co-ordinately at the level of mRNA.Abbreviations poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Epoxiconazole-induced stimulation of the antifungal hydrolases chitinase and β-1,3-glucanase in wheat

Young plants of wheat (Triticum aestivum L. cv. Star), which were treated hydroponically with the triazole fungicide epoxiconazole (BAS 480 F) over a period of 8 days, showed a dose-dependent stimulation of the enzyme activities of the two antifungal hydrolases chitinase and -1,3-glucanase in the shoot tissue. In the root tissue, no significant rise in the enzyme activities was found. As shown by immunoblot analysis and enzyme-linked immunosorbent assay (ELISA) using antisera against tobacco acidic and basic chitinases and -1,3-glucanases, the obeserved increase in the activities coincided with an accumulation of enzyme proteins. This possibly indicates the induction of a de novo synthesis of chitinases and -1,3-glucanases by epoxiconazole. To our knowledge, this effect of a synthetic fungicide on antifungal hydrolases in an intact plant is demonstrated for the first time.     

Characterization and expression of chitinase and 1,3-β-glucanase genes in cotton

We have isolated cDNA clones representing mRNAs encoding chitinase and 1,3--glucanase in cotton (Gossypium hirsutum L.) leaves. The chitinase clones were sequenced and found to encode a 28,806 Da protein with 71% amino acid sequence similarity to the SK2 chitinase from potato (Solanum tuberosum). The 1,3--glucanase clones encoded a 37,645 Da protein with 57.6% identity to a 1,3--glucanase from soybean (Glycine max). Northern blot analyses showed that chitinase mRNA is induced in plants treated with ethaphon or salicylic acid, whereas the levels of 1,3--glucanase mRNA are relatively unaffected. Southern blots of cotton genomic DNA and genomic clones indicated chitinase is encoded by a small gene family of which two members, Chi 2;1 and Chi 2;2, were characterized. These genes share 97% sequence identity in their transcribed regions. The genes were found to have three exons which are 309, 154 and 550 bp long, and two introns 99 and 154 bp in length. The 5-flanking regions of Chi 2;1 and Chi 2;2 exhibit a large degree of similarity and may contain sequences important for gene response to chemical agents and fungal attack.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β-triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.     

The effect of ethylene on the cell-type-specific and intracellular localization of β-1,3-glucanase and chitinase in tobacco leaves

We have studied the effect of ethylene on the localization of the basic isoforms of glucan endo-1,3--glucosidase (-1,3-glucanase, EC 3.2.1.39) and endo-chitinase (chitinase, EC 3.2.1.14) in leaves of Nicotiana tabacum L. cv. Havana 425. Comparisons of the enzyme contents of the lower epidermis of the leaf, leaf expiants with the lower epidermis removed, and intercellular wash fluid indicate that both enzymes are localized inside epidermal cells of untreated leaves. Ethylene treatment (20 l·l^-1, 4d) induced a marked -10- to 30-fold-coordinated accumulation of the enzymes. This was due primarily to induction of the basic isoforms inside chlorenchyma cells of the leaf interior. The localization of basic -1,3-glucanase was confirmed by immunofluorescence histochemistry and immunogold cytochemistry. Immunolabelling was confined to electron-dense bodies of the cell vacuole. No extracellular immunolabelling was detected in control or ethylene-treated leaves. We conclude that ethylene changes the cell-type-specific distribution but not the intracellular compartmentation of the two enzymes. These results support the generalization that basic isoforms of chitinase and -1,3-glucanase are intracellular whereas the acidic isoforms are secreted into the extracellular space.Abbreviations IgG immunoglobulin G - IWF intercellular wash fluid - PBS 0.14 M NaCl, 0.1 M K₂HPO₄, pH 7.5 - TMV tobacco mosaic virus We thank Monique Seldran and Alfred Milani for expert technical help, Patricia Ahl-Goy, Ciba-Geigy, AG, Basel for supplying IWF from TMV-infected leaves, and our colleagues Thomas Boller and Lilian Sticher for their comments and criticism.     

Biotransformation of dehydroepiandrosterone with Macrophomina phaseolina and β-glucuronidase inhibitory activity of transformed products

The biotransformation of dehydroepiandrosterone (1) with Macrophomina phaseolina was investigated. A total of eight metabolites were obtained which were characterized as androstane-3,17-dione (2), androst-4-ene-3,17-dione (3), androst-4-ene-17β-ol-3-one (4), androst-4,6-diene-17β-ol-3-one (5), androst-5-ene-3β,17β-diol (6), androst-4-ene-3β-ol-6,17-dione (7), androst-4-ene-3β,7β,17β?triol (8), and androst-5-ene-3β,7α,17β-triol (9). All the transformed products were screened for enzyme inhibition, among which four were found to inhibit the β-glucuronidase enzyme, while none inhibited the α-chymotrypsin enzyme.     

Characterization and expression of β-1,3-glucanase genes in peach

Beta-1,3-glucanase is one of the pathogenesis-related (PR) proteins involved in plant defense responses. A peach beta-1,3-glucanase gene, designated PpGns1, has been isolated and characterized. The deduced amino acid sequence of the product of PpGns indicates that it is a basic isoform (pI 9.8), and contains a putative signal peptide of 38 amino acids but has no C-terminal extension. Amino acid sequence comparisons revealed that PpGns1 is 69% and 67% identical to citrus and soybean beta-1,3-glucanases, respectively. Southern analysis of total genomic DNA also indicates that at least three genes for beta-1,3-glucanases exist in peach, forming a small gene family. Characterization of four additional clones by PCR has identified a second beta-1,3-glucanase gene, PpGns2. PpGns2 has been partially sequenced, and when compared to PpGns1, it shows high sequence homology, 96% and 99% nucleotide identity in the first and (partial) second exons, respectively. The deduced partial sequence of the PpGns2 product displays only two differences from PpGns1 in the signal peptide and one in the (partial) mature protein (141 amino acids). The 5'-flanking promoter regions of these two genes share 90% identity in nucleotide sequences interrupted by five major gaps (4-109 nt long). The promoter region contains various sequences similar to cis-regulatory elements present in different stress-induced plant genes. In leaves and stems of peach shoot cultures grown in vitro, PpGns1 is induced within 12 h after exposure to a culture filtrate of Xanthomonas campestris pv. pruni or ethephon. However, it is not induced following treatment with mercuric chloride.     

Developmental and hormonal regulation of β-1,3-glucanase in tobacco

A highly sensitive and specific rocket immunoassay was used to measure the content of an endo-type -1,3-glucanase (EC 3.2.1.39) in tissues of Nicotiana tabacum L. cv. Havana 425. We show that the accumulation of -1,3-glucanase in cultured pith-parenchyma tissue is blocked by combinations of the auxin, -naphthaleneacetic acid (NAA), and the cytokinin, kinetin. When tissues pre-incubated for 7 d on complete medium containing 2.0 mg·l^-1 NAA and 0.3 mg·l^-1 kinetin are transferred onto medium without hormones or with either hormone added separately, the -1,3-glucanase content expressed per mg soluble protein increases approx. ten fold over a 7-d period. Under these inductive conditions, up to approx. 5% of the soluble protein is -1,3-glucanase. The induction is inhibited by >90% when tissues are cultured over the same period on medium containing both hormones. This -1,3-glucanase is developmentally regulated in the intact plant. It is a major component of the soluble protien in the lower leaves and roots but is not detectable in leaves near the top of the plant.Abbreviation NAA -naphthaleneacetic acid     

Ethylene-induced chitinase and β-1,3-glucanase accumulate specifically in the lower epidermis and along vascular strands of bean leaves

We have studied the spatial pattern of accumulation of chitinase (EC 3.2.1.14) and -1,3-glucanase (EC 3.2.1.39) in ethylene-treated leaves of bean (Phaseolus vulgaris L.). Electron-microscopical examination of chemically fixed tissue demonstrated the presence of large electron-dense aggregates in the vacuoles of ethylene-treated leaf cells. No such vacuolar structures were observed in untreated control cells. Immunogold labelling with antisera directed against the basic forms of chitinase and -1,3-glucanase indicated that the vacuolar aggregates were the major site of accumulation of chitinase and -1,3-glucanase. The chitinase- and -1,3-glucanase-containing vacuolar aggregates were not randomly distributed within the leaf tissue but were restricted to the lower epidermal cells and to parenchyma cells adjacent to vascular strands. In addition, heavy -1,3-glucanase labelling was observed over spongy plugs of expanded middle-lamella material that appear to occlude the transition regions between the airspaces underlying the stomata and those throughout the rest of the leaf. Some labelling was also seen to extend along the surface layer of the cell walls lining all of the airspaces. Protein analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting as well as enzyme-activity measurements showed that the peeled lower epidermis of the ethylene-treated leaves contained on a protein and on a per-weight basis several times more chitinase and -1,3-glucanase than the remainder of the leaf.Abbreviation in Text SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Abbreviations in Micrographs AS air space - C chloroplast - EP (epidermal) cell - G guard cell - P parenchyma cell - S stoma - V vacuole - VE] vein - VP vascular parenchyma cell - W cell wall - X xylem We thank Dr. L.A. Hadwiger, Pullman, Wash., and Dr. U. Vögeli, Lexington, Ky., for their kind gifts of antibodies. This work was supported by the National Science Foundation grant DCB-8615763 to L.A.S.     

Primary structure and expression of mRNAs encoding basic chitinase and 1,3-β-glucanase in potato

Infection of potato leaves (Solanum tuberosum L. cv. Datura) by the late blight fungus Phytophthora infestans, or treatment with fungal elicitor leads to a strong increase in chitinase and 1,3--glucanase activities. Both enzymes have been implicated in the plant's defence against potential pathogens. In an effort to characterize the corresponding genes, we isolated complementary DNAs encoding the basic forms (class I) of both chitinase and 1,3--glucanase, which are the most abundant isoforms in infected leaves. Sequence analysis revealed that at least four genes each are expressed in elicitor-treated leaves. The structural features of the potato chitinases include a hydrophobic signal peptide at the N-terminus, a hevein domain which is characteristic of class I chitinases, a proline- and glycine-rich linker region which varies among all potato chitinases, a catalytic domain, and a C-terminal extension. The potato 1,3--glucanases also contain a N-terminal hydrophobic signal peptide and a C-terminal extension, the latter comprising a potential glycosylation site. RNA blot hybridization experiments showed that basic chitinase and 1,3--glucanase are strongly and coordinately induced in leaves in response to infection, elicitor treatment, ethylene treatment, or wounding. In addition to their activation by stress, both types of genes are regulated by endogenous factors in a developmental and organ-specific manner. Appreciable amounts of chitinase and 1,3--glucanase mRNAs were found in old leaves, stems, and roots, as well as in sepals of healthy, untreated plants, whereas tubers, root tips, and all other flower organs (petals, stamen, carpels) contained very low levels of both mRNAs. In young leaves and stems, chitinase and 1,3--glucanase were differentially expressed. While chitinase mRNA was abundant in these parts of the plant, 1,3--glucanase mRNA was absent. DNA blot analysis indicated that in potato, chitinase and 1,3--glucanase are encoded by gene families of considerable complexity.     

Localization of β-1,3-glucanase in Trichoderma harzianum

Studies on the constitutive β-1,3-glucanase were conducted in submerged as well as in the stationary culture conditions, in the presence and in the absence of lactose and glucose as main carbon sources. In the absence of lactose or glucose, expression of β-1,3-glucanase was observed at 96?h in extracellular, periplasmic, cell wall bound and internal fractions during submerged fermentation. In shake flask culture, enzyme was found in all subcellular fractions using optimal glucose concentration. When Trichoderma harzianum was grown on media containing 55?kg lactose/m³ in submerged culture, activity was found in extracellular, cell wall bound and in the periplasmic fractions. The relative distribution of the enzyme in the cell is independent of the nature of the carbon source and its concentration.     

Extracellular targeting of the vacuolar tobacco proteins AP24, chitinase and β-1,3-glucanase in transgenic plants

The Nicotiana tabacum ap24 gene encoding a protein with antifungal activity toward Phytophthora infestans has been characterized. Analysis of cDNA clones revealed that at least three ap24-like genes are induced in tobacco upon infection with tobacco mosaic virus. Amino acid sequencing of the purified protein showed that AP24 is synthesized as a preproprotein from which an amino-terminal signal peptide and a carboxyl-terminal propeptide (CTPP) are cleaved off during post-translational processing. The functional role of the CTPP was investigated by expressing chimeric genes encoding either wild-type AP24 or a mutant protein lacking the CTPP. Plants expressing the wild-type construct resulted in proteins properly sorted to the vacuole. In contrast, the proteins produced in plants expressing the mutant construct were secreted extracellularly, indicating that the CTPP is necessary for targeting of AP24 to the vacuoles. Similar results were obtained for vacuolar chitinases and -1,3-glucanases of tobacco. The extracellularly targeted mutant proteins were shown to have retained their biological activity. Together, these results suggest that within all vacuolar pathogenesis-related proteins the targeting information resides in a short carboxyl-terminal propeptide which is removed during or after transport to the plant vacuole.     

Stability of transgene integration and expression in subsequent generations of doubled haploid oilseed rape transformed with chitinase and β-1,3-glucanase genes in a double-gene construct

A double-gene construct with one chitinase and one β-1,3-glucanase gene from barley, both driven by enhanced 35S promoters, was transformed into oilseed rape. From six primary transformants expressing both transgenes 10 doubled haploid lines were produced and studied for five generations. The number of inserted copies for both the genes was determined by Southern blotting and real-time PCR with full agreement between the two methods. When copy numbers were analysed in different generations, discrepancies were found, indicating that at least part of the inserted sequences were lost in one of the alleles of some doubled haploids. Chitinase and β-1,3-glucanase expression was analysed by Western blotting in all five doubled haploid generations. Despite that both the genes were present on the same T-DNA and directed by the same promoter their expression pattern between generations was different. The β-1,3-glucanase was expressed at high and stable levels in all generations, while the chitinase displayed lower expression that varied between generations. The transgenic plants did not show any major impact on fungal resistance when assayed in greenhouse, although purified β-1,3-glucanase and chitinase caused retardment of fungal growth in vitro.     

1,3-β-Glucanase from Vigna aconitifolia and its possible use in enzyme bioreactor fabrication

Endo-1,3(4)-β-glucanase (EC 3.2.1.6) from Vigna aconitifolia sprouts was purified to 14.5 fold by gel filtration and ion-exchange chromatography. The enzyme was found to be a glycoprotein, its activity was Ca²⁺ dependent and specific for β-1,3 linkages in different polysaccharides. The K_m value of the enzyme was estimated to be 3.0 mg ml⁻¹ for β-d-glucan as substrate. Circular dichroism studies revealed 8% α-helix, 48% β-pleated and 44% random coil in its secondary structure. Purified β-glucanase was then successfully co-immobilized with glucose oxidase in agarose-chitosan beads, showing better immobilization yield, operational range and stability as compared with the crude β-glucanase beads. The immobilized β-glucanase was successfully used for mini-bioreactor fabrication.     

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.     

Role of β-1,3-glucanase in postmeiotic microspore release

Stage-specific extracts of Lilium anthers undergoing meiosis exhibited sharp peaks of both endolytic and exolytic β-1,3-glucanase activity at the time of in situ callose breakdown. The endo- and exo-β-1,3-glucanase activities, attributable to different enzymes, were found to have molecular weights of 32,000 and 62,000, respectively. The majority of exoglucanase activity was found in the outer somatic layers of the anther, whereas the majority of endoglucanase activity was located in the immediate surroundings of the meiocytes. The action of both glucanase activities on callose wall removal was monitored. It was shown that endo-β-1,3-glucanase, but not exoglucanase, was able to effect callose wall removal. To the extent that detection of glucanase activity in extracts reflects its activity in vivo, the endoglucanase enzyme may be considered as the immediate agent of callose wall breakdown and, hence, as a critical regulator in the initiation of the development of the gametophyte stage.     

Chitinase and β-1,3-glucanase in the lutoid-body fraction of Hevea latex

The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are, besides the chitin-binding protein hevein, its precursor and the C-terminal fragment of this precursor, proteins with enzymic activities: three hevamine components, which are basic, vacuolar, chitinases with lysozyme activity, and a β-1,3-glucanase. Lutoid-body fractions from three rubber-tree clones differed in their contents of these enzyme proteins. The hevamine components and glucanase were isolated and several enzymic and structural properties were investigated. These enzymes are basic proteins and cause coagulation of the negatively charged rubber particles. The coagulation occurs in a rather narrow range of ratios of added protein to rubber particles, which indicates that charge neutralization is the determining factor. Differences in coagulation of rubber particles by lutoid-body fractions from various rubber clones can be explained by their content of hevamine and glucanase. Glucanase from the lutoid-body fraction may dissolve callus tissue and this may explain the observation that rubber-tree clones with a high glucanase content in this fraction produce more latex than clones with little glucanase. Sequence studies of two CNBr peptides of the glucanase indicate that this protein is homologous with glucanases from other plants, and that a C-terminal peptide, possibly involved in vacuolar targeting, may have been cleaved off.     

Ozone-induced changes of mRNA levels of β-1,3-glucanase,chitinase and ‘pathogenesis-related’ protein 1b in tobacco plants

Treatment of the ozone-sensitive tobacco cultivar Bel W3 with an ozone pulse (0.15 l/l, 5 h) markedly increased the mRNA level of basic -1,3-glucanase and to a lower degree that of basic chitinase. The increase of -1,3-glucanase mRNA level occurred within 1 h and showed a transient maximum. Seventeen hours after ozone treatment, the -1,3-glucanase mRNA level decreased to lower values. The increase of basic chitinase mRNA level was delayed and was less pronounced than that of -1,3-glucanase mRNA. Cultivar Bel B showed only a small increase of -1,3-glucanase mRNA level after the same ozone treatment, whereas its basic chitinase mRNA was more strongly induced. Prolonged ozone treatment for 2 days of tobacco Bel W3 led to a persistent level of -1,3-glucanase and basic chitinase mRNAs, as well as to an increase of acidic chitinase and pathogenesis-related (PR) 1b mRNA levels. The results indicate that genes so far considered to code for PR proteins may also be involved in the plant response to oxidative stress.