Cellulase activity and localization during induced abscission of Coleus blumei

Treatment with dimethipin (2,3-dihydro-5,6-dimethyl-1,4-dithiin 1,1,4,4 tetroxide) inhibited the increase in cellulase activity and decrease in breakstrength associated with the normal course of abscission in Coleus. Application of the surfactant UBI-1126 (Emery OAL 20 in isopropyl alcohol) increased cellulase activity and accelerated the process of abscission in Coleus expiants within 24 h of application. Cellulase activity was localized histochemically at the electron microscopic level in surfactant-treated tissue. The enzyme activity was localized primarily in the cell wall, middle lamella, and paramural bodies of abscission zone cells.     

Endo‐1,4‐β‐glucanase gene expression and cell wall hydrolase activities during abscission in Valencia orange

The physiological and molecular events of ethylene‐induced abscission in mature fruit calyx, laminar and floral abscission zones of cv. Valencia orange were examined. Continuous exposure of fruit explants to 5 µl 1⁻¹ ethylene for 2 to 40 h resulted in marked increases in endo‐1,4‐β‐glucanase (cellulase) and polygalacturonase (PG) activities in calyx abscission zones. Two abscission‐related cellulases and one PG were found. The major peak of cellulase activity corresponded to a pI of 8.0 and molecular weight of 51 kDa, whereas the minor cellulase peak had a pI of 5.5. The abscission polygalacturonase had a pI of 5.5. Calyx abscission zone RNA was amplified with degenerate primers based on sequence of the purified Valencia orange calyx abscission cellulase, and cloned. The two partial cellulase cDNA clones were 59% identical at the nucleotide level. Genomic Southern analysis suggested that Valencia orange contained two groups of cellulase genes. A full‐length cDNA clone from each group was isolated from a cDNA library prepared from ethylene‐induced calyx abscission zone mRNA. Both genes were expressed in ethylene‐induced calyx, laminar and floral abscission zones, but were not expressed in non‐induced abscission zones or mature leaves treated with or without ethylene, young bark or young fruit of Valencia.     

Exoglucanases fromZea mays L. seedlings: their role inβ-D-glucan hydrolysis and their potential role in extension growth

Exoglucanases of corn seedlings were examined and evaluated in terms of their participation in the hydrolysis of cell-wall β-D-glucan and their possible role in extension growth. An exo-β-1,3-glucanase (EC 3.2.1.58), a component of the protein dissociated from isolated wall by use of high salt solutions, was purified using gel-filtration and ion-exchange chromatography. The purified enzyme hydrolyzed a number of polymeric and oligosaccharide substrates, including those of mixedlinkage, and their direct conversion to monosaccharide was evidence that the enzyme was capable of hydrolyzing both β1–4 and β1–3 linkages. The enzyme was considerably more active toward glucan that had been previously hydrolyzed by a cell-wall endo-β-D-glucanase. Similarly, the capacity of the purified exo-β-D-glucanase to degrade isolated wall was enhanced by more than 60% when the wall had been previously treated with the endoenzyme. The exo-β-D-glucanase did not exhibit growth-promoting properties nor was its activity, measured in vivo, enhanced by auxin. Another glucanase was obtained from the soluble fraction of seedling homogenates. It functioned strictly as a β-glucosidase and did not appear to participate in the hydrolysis of wall β-D-glucan.     

Purification and characterization of thermostable β-1,3-1,4 glucanase from<Emphasis Type="Italic">Bacillus</Emphasis> sp. A8-8

In this study, the extracellular enzyme activity ofBacillus sp. A8-8 was detected on LB agar plates containing 0.5% of the following substrates: carboxymethylcellulose (CMC), xylan, cellulose, and casein, respectively. The β-1,3-1,4 glucanase produced fromBacillus sp. A8-8 was purified by ammonium sulfate and hydrophobic chromatography. The molecular size of the protein was estimated by SDS-PAGE as approximately 33 kDa. The optimum pH and temperature for the enzyme activity were 6.0 and 60°C, respectiveley. However, enzyme activity was shown over a broad range of pH values and temperatures. The purified β-1,3-1,4 glucanase retained over 70% of its original activity after incubation at 80°C for 2 h, and showed over 40% of its original activity within the pH range of 9 to 12. This suggests that β-1,3-1,4 glucanase fromBacillus sp. A8-8 is thermostable and alkalistable. In addition, β-1,3-1,4 glucanase had higher substrate specificity to lichenan than to CMC. Finally the activity of the endoglucanase was inhibited by Fe³⁺, Mg²⁺, and Mn²⁺ ions. However Co²⁺ and Ca²⁺ ions were increased its activity. These authors contributed equally to this work.     

Lytic β-1,3 Glucanase from Arthrobacter: Pattern of Action

The action pattern of lytic β-1,3 glucanase (glucanase I) from Arthrobacter which liberates predominantly laminaripentaose from various β-glucans has been studied. The enzyme was not active on short linear laminaridextrins, but was active on an enzymatically synthesized, linear β-1,3 oligoglucan preparation. Any intactness of the glucose residues of the chain ends of a substrate did not seem to be necessary for the action of the enzyme. The results of determination of laminaripentaose during a relatively early phase of the reaction suggested that about half of the reducing power liberated in the medium might be explained by the formation of the sugar. It seems that the formation of laminaripentaose relates to the initial attack of glucanase 1 on β-1,3 glucan chains.     

Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH

In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0–5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed.     

A Role for the Stele in Intertissue Signaling in the Initiation of Abscission in Bean Leaves (Phaseolus vulgaris L.)

A combination of microdissection and viscometric endo-[beta]-1,4-glucanhydrolase assays was used to investigate if the early appearance of the abscission-related isoelectric point-9.5 endo-[beta]-1,4-glucanhydrolase in the stele of the pulvinus and abscission zone of the foliar abscission zone of Phaseolus vulgaris L. prior to cell separation (reported by E. del Campillo, P.D. Reid, R. Sexton, L.N.Lewis [1990] Plant Cell 2: 245-254) indicates that the vascular tissue of this region has a specific role in abscission. We find that no endo-[beta]-1,4-glucanhydrolase activity or cell separation is detectable in the abscission zone cortex if the abscission zone cortex is separated from the stele tissue. If the stele is separated from the abscission zone cortex after a lag period but again before any endo-[beta]-1,4-glucanhydrolase activity is present in the abscission zone cortex, then the enzyme is produced in the cortex and abscission ensues. We conclude that the cortex of the abscission zone is able to abscind independently of the vascular tissue only after the vascular tissue has begun to respond to abscission-promoting signals. We suggest that ethylene promotes formation of an abscission-permitting signal in the stele of the abscission zone and pulvinus, and that this signal is an essential elicitor for the synthesis of cell separation enzymes in the target cells of the abscission zone cortex.     

The use of immunological methods to study the activity of cellulase isozymes (B 1:4 glucan 4-glucan hydrolase) in bean leaf abscission

Abstract The changes in the levels of two different isozymes of cellulase (EC 3.2.1.4) have been followed during the abscission of the primary leaves of bean (Phaseolus vulgaris c.v. Red Kidney), using antibodies raised against the 9.5 form of the enzyme. Data from both radioimmune and direct assay show that the 9.5 form of cellulase is undetectable prior to the induction of abscission. After a 12 h lag this isozyme increases in activity, the increase preceding a decrease in integrity of the abscission zone cell walls. The results are consistent not only with the view that this specific isozyme is involved in wall hydrolysis but also with previous data which showed that cellulase is synthesized ‘de novo’. The 4.5 isozyme of cellulase is more widely spread throughout the plant, being most active in young tissues. During abscission the activity of this isozyme in the abscission layer falls and consequently it is not thought to be involved directly in the abscission process.     

An ultrastructural study of abscission zone cells with special reference to the mechanism of enzyme secretion

Summary The ultrastructural changes which occur during the foliar abscission ofImpatiens sultani Hook. andColeus blumei Benth. have been described. In both cases fracture of the separation zone results from a modification of the wall and cleavage along the line of the middle lamella. This process starts at the corners of the cells and in regions rich in plasmodesmata.During the period of wall breakdown, cellular integrity is maintained and the membrane degradation described by others was not observed. Plasmolysis studies confirmed that the separation zone cells retained their selective permeability characteristics until well after wall fracture. Quantitative data are presented to show that there is an increase in the frequency of rough endoplasmic reticulum, dictyosomes and dictyosome vesicles during the period when wall-hydrolyzing enzyme secretion is occurring. These changes are interpreted as reflecting an increase in demand for the secretory machinery of the endomembrane system. The possible involvement of plasmodesmata in this process is also discussed.     

Purification and characterization of beta-1,6-glucanase of Streptomyces rochei application in the study of yeast cell wall proteins

A beta-1,6-glucanase was purified to apparent homogeneity from a commercial yeast digestive enzyme prepared from Streptomyces rochei by a series of column chromatographies. The molecular mass of the purified enzyme was 60 kDa by SDS-PAGE. The purified enzyme had an optimum pH range from 4.0 to 6.0 and was stable in the same pH range. The enzyme was stable under 50 degrees C but lost almost all activity at 60 degrees C. The enzyme was specific to beta-1,6-glucan and had little activity towards beta-1,3-glucan and beta-1,4-glucan. When the beta-1,6-glucan was hydrolyzed with the purified enzyme for 5 h, the reaction products contained 20% glucose, 36% gentiobiose, and 44% other oligosaccharides, suggesting that the enzyme is an endo-type glucanase. When the purified enzyme was used for the digestion of the cell wall of Saccharomyces cerevisiae, cell-wall proteins covalently bound to the cell-wall glucan were recovered as soluble forms, suggesting that this enzyme is useful for analysis of yeast-cell wall proteins.     

Anatomical Changes and Immunolocalization of Cellulase during Abscission as Observed on Nitrocellulose Tissue Prints

A fundamental event in abscission is the breakdown of cell wall material in a discrete zone of cells known as the separation layer. Three dimensional images produced by viewing tissue prints of abscission zones on nitrocellulose (NC) membranes with incident illumination showed changes in the tissue integrity taking place in the separation layer as the process of abscission proceeded. The cell softening which occurs due to the dissolution of the cell wall appeared in the tissue prints as a diffuse line at the anatomical transition between the pulvinus and petiole and was easily observed on NC tissue prints of either longitudinal or serial cross-sections through abscission zones. In bean leaf abscission the dissolution of cell walls has been correlated with the appearance of a form of cellulase with an isoelectric point of pH 9.5. Antibodies specific for this enzyme were used to study the localization of 9.5 cellulase in the distal abscission zone of Phaseolus vulgaris L., cv Red Kidney after tissue printing on NC. It was found that 9.5 cellulase was localized in the separation layer but also occurred in the vascular tissue of the adjacent pulvinus. No antibody binding was observed in nonabscising tissue or preimmune controls. These results confirm previous biochemical studies and demonstrate that immunostaining of nitrocellulose tissue prints is a fast and reliable method to localize proteins or enzymes in plant tissue.     

EVIDENCE FOR GLUCAN COMPONENTS IN THE PRIMARY ZYGOTE WALL OF CHLAMYDOMONAS MONOICA

The primary zygote wall of C. monoica is transient and is released from mature zygospores. The fluorochromes aniline blue and primulin, used in other systems to detect β-1,3 glucans, stain the primary wall intensely. Two β-1,3 glucan synthases have been identified in higher plants: a calcium-dependent synthase produced in response to wounding and induced by chitosan, and a magnesium-dependent enzyme, associated with pollen development and unresponsive to chitosan. Chitosan has no effect on C. monoica primary wall synthesis or staining properties. We are presently testing for the effect of magnesium and/or calcium depletion on primary wall synthesis. Aniline blue and primulin do not stain purified cellulose fibers, while the fluorochrome Calcofluor does. Calcofluor also stains the primary wall intensely. For all fluorochormes tested, fluorescence is first detected in motile quadriflagellate zygotes. Aniline blue staining maximizes quickly, while Calcofluor staining continues to intensify until primary wall release. Dinitrobenzonitrile, a specific inhibitor of cellulose synthesis in plants, has no effect on primary wall synthesis in C. monoica. Addition of glucanase or cellulase to partially purified primary walls results in wall thinning and loss of staining. Using electron microscopy, we are evaluating the effects of these enzymes on primary wall ultrastructure. Further studies are needed to determine whether all three fluorochromes are recognizing the same polysaccharide component (a β-1,3 glucan or a β-1,3; β-1,4 mixed glucan), or whether Calcofluor staining indicates the presence of a distinct component containing β-1,4 linkages, such as cellulose or a xyloglucan.     

beta-d-Glucan of Avena Coleoptile Cell Walls

A specific glucanase was used to liberate a noncellulosic beta-d-glucan from isolated cell walls of Avena sativa coleoptile tissue. Cell walls of this tissue contain as much as 7 to 9 mg of glucan/100 mg of dry wall. Because of the specific action pattern of the enzyme, a linkage sequence of.. 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc.. is indicated and the predominance of trisaccharide and tetrasaccharide as hydrolytic products suggests a rather regular repeating pattern in the polysaccharide. The trisaccharide and the tetrasaccharide are tentatively identified as 3-O-beta-cellobiosyl-d-glucose and 3-O-beta-cellotriosyl-d-glucose, respectively. Recovery of these oligosaccharides following glucanase treatment of native wall material was feasible only after wall-bound glucosidases were inactivated. In the absence of enzyme inactivation the released fragments were recovered as glucose. The beta-d-glucan was not extracted from walls by either hot water or protease treatment.Cell walls prepared from auxin-treated Avena coleoptile segments yielded less glucan than did segments incubated in buffer suggesting an auxin effect on the quantity of this wall component. No IAA-induced change in the ratio of the trisaccharide and tetrasaccharide could be detected, suggesting no shift in the 1,3 to 1,4 linkage ratio. While the enzyme acts directly on the beta-d-glucan, no elongation response was apparent when Avena sections were treated with the purified glucanase. The presence of the glucan was not associated with any wound response which could be attributed to the preparation of coleoptile segments. The relationship of glucan metabolism to auxin growth responses is discussed.     

Purification and Properties of Lytic β-Glucanase from an Arthrobacter Bacterium

A glucanase was isolated from a culture fluid of an Arthrobacter bacterium. The purified enzyme preparations consisted of the glucanase components having the same enzymatic activity. The enzyme was stable in a broad pH range, but lost its activity rapidly at above 60°C. Optimum pH values were found to be 5.5~6.5.

The glucanase attacked the following glucan preparations and liberated a relatively small amount of reducing power: Saccharomyces cerevisiae glucan, Candida albicans glucan, Saccharomyces fragilis glucan, pachyman, curdlan and laminaran. The most prominent sugar spot on the chromatogram of the digest from yeast glucan was identified with laminan-pentaose, and the other faint spots with a series of laminaridextrins. The β-1,6 glucosidic bonds in yeast glucan were not hydrolyzed and concentrated in a soluble fraction which was found near the origin of the chromatogram.     

Some ultrastructural observations on the nature of foliar abscission in Impatiens sultani

Summary Both scanning and transmission electron microscopes have been used to study the anatomy of the abscission zone of Impatiens sultani Hook. Evidence is presented to show that the fracture line follows the middle lamella in all the living cells of the abscission zone including those in the vascular traces. The separation of these cells is preceded by a breakdown of the middle lamellar region of the wall. The characteristics of this process vary in different cell types. Accompanying this breakdown is an enlargement of inner cortex cells mainly in a direction parallel to the axis of the petiole. It is suggested that this expansion of cells is necessary to produce the tensions which rupture the cuticle and xylem vessels prior to separation. The occurrence of transfer cells and tyloses in the abscission zone is also described and the physiological implications of their presence discussed.     

Spatial and temporal aspects of cell separation in the foliar abscission zones ofImpatiens sultani Hook

Summary The abscission of leaves fromImpatiens sultani Hook. occurs as the direct result of the weakening of a narrow band of cells running transversely across the base of the petiole. This loss of strength of the abscission zone is due to the breakdown of the central cell wall in two or three layers of cells. The process of wall degeneration is first visible 13 hours after the induction of abscission in a small group of cells found just below the concave groove on the adaxial side of the petiole. As the abscission zone gets progressively weaker the area of cells showing wall breakdown expands, spreading through the parenchyma to the lower side of the stele. The walls of the collenchyma and epidermis along the sides and base of the petiole and the central vascular tissues are the last to break down. Experiments in which the abscission zone was dissected into small pieces were undertaken to investigate whether cell wall hydrolysis was a contagious phenomenon, spreading from cell to cell by direct contact. The results of these investigations indicated that there was little requirement for cell to cell contact in either the temporal or spatial integration of cell wall breakdown.     

Exo- and endoglucanases of maize coleoptile cell walls: their interaction and possible regulation

Glucanase-mediated degradation of beta-(1,3)(1,4)-glucans has been attributed to auxin-induced cell wall loosening and thus tissue growth in cereal plants, but the regulatory mechanisms for the auxin-enhancement of glucanase activities in situ are not fully understood. Here, we report evidence for possible mechanisms which might account for auxin-induced changes in glucanase activities. A likely cause for acceleration of wall glucan degradation is the change in the ratio of exo- and endoglucanases. The combined enzymes synergistically promote beta-(1,3)(1,4)-glucan hydrolysis. In addition, these enzyme activities are enhanced by other enzymic and non-enzymic proteins and are also partially stimulated by divalent cations such as Ca(2+) and Mg(2+) at certain pH values. The acceleration of glucan degradation mediated by auxin may be mediated by changes and/or interactions of any of these factors in situ.     

Immunolocalization of 1,3‐β‐Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

The distribution of extracellular 1,3‐β‐glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3‐β‐glucanase secreted by the pathogenic fungus. A specific antibody of 1,3‐β‐glucanase, anti‐GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3‐β‐glucanase of Ggt, but not for 1,3‐β‐glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3‐β‐glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen‐infected wheat roots with anti‐1,3‐β‐glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non‐inoculated wheat roots. The results indicate that 1,3‐β‐glucanase secreted by Ggt may be involved in pathogenesis of the take‐all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.