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.     

beta-d-Glucan Hydrolase Activity in Zea Coleoptile Cell Walls

Enzymes dissociated from corn (hybrid B73 x Mo17) seedling cell walls by solutions of high ionic strength possess the capacity to degrade Avena caryopsis glucan. Inhibitor studies disclosed that both endo- and exoenzyme activities were involved and that the reaction sequence paralleled the autolytic solubilization of beta-d-glucan in isolated cell walls.The salt-dissociated exoenzyme activity was strongly inhibited by HgCl(2) and to a lesser extent by parachloromercuribenzoate at a concentration of 100 micromolar. In the absence of these inhibitors, Avena caryopsis glucan was converted to monosaccharide, whereas in the presence of the mercurials, only endoenzyme activity was apparent and the glucan substrate was hydrolyzed yielding products with an average molecular size of 1.5 to 3.0 x 10(4) daltons. Endoenzyme hydrolysis of the caryopsis glucan could not be attributed to the participation of an enzyme specific for mixed-linkage substrates.The autolytic capacity of isolated cell walls was similarly affected by inhibitors. In the presence of 100 micromolar HgCl(2), cell walls released from 60 to 80 micrograms per milligram dry weight as polymeric glucan during a 24-hour period. Monosaccharide accounted for less than 2% of the autolytically solubilized products. Analysis of the polymeric glucan product revealed a similarity in molecular size to the products obtained following treatment of Avena caryopsis glucan with salt-dissociated wall protein. The results suggest that among the salt-dissociated proteins are those responsible for the autolytic capacity of isolated cell walls.     

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.     

Partial purification of cell wall α-galactosidases and α-arabinosidases from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes

Two protein fractions with activity as α-galactosidase (EC 3.2.1.22) and α-arabinosidase (EC 3.2.1.55), respectively, were identified in the proteins of cell wall of Cicer arietinum L. cv. Castellana extracted with 3 M LiCl. These fractions were partially purified by gel filtration chromatography (Bio Gel P-150), increasing the specific arabinosidase activity 57-fold and the α-galactosidase activity 6-fold. Other protein fractions with glucosidase (EC 3.2.1.21) and glucanase (EC 3.2.1.6) activity also appeared. According to earlier authors, α-arabinosidases and α-galactosidases are related to alterations in linkages occurring in cell walls, since the enzymes are able to hydrolyze isolated wall polymers. However, our preparations hydrolyze intact cell walls only to a very limited extent, such that their participation in the autolytic processes of cell walls can be ruled out.     

Secondary cell wall formation in Cryptococcus neoformans as a rescue mechanism against acid-induced autolysis

Growth of the opportunistic yeast pathogen Cryptococcus neoformans in a synthetic medium containing yeast nitrogen base and 1.0–3.0% glucose is accompanied by spontaneous acidification of the medium, with its pH decreasing from the initial 5.5 to around 2.5 in the stationary phase. During the transition from the late exponential to the stationary phase of growth, many cells died as a consequence of autolytic erosion of their cell walls. Simultaneously, there was an increase in an ecto-glucanase active towards β-1,3-glucan and having a pH optimum between pH 3.0 and 3.5. As a response to cell wall degradation, some cells developed an unusual survival strategy by forming 'secondary' cell walls underneath the original ones. Electron microscopy revealed that the secondary cell walls were thicker than the primary ones, exposing bundles of polysaccharide microfibrils only partially masked by an amorphous cell wall matrix on their surfaces. The cells bearing secondary cell walls had a three to five times higher content of the alkali-insoluble cell wall polysaccharides glucan and chitin, and their chitin/glucan ratio was about twofold higher than in cells from the logarithmic phase of growth. The cell lysis and the formation of the secondary cell walls could be suppressed by buffering the growth medium between pH 4.5 and 6.5.     

Effects of glucanase treatment on the structure and mechanical properties of cell walls in the giant‐celled xanthophycean alga Vaucheria frigida

The cell wall of the tip‐growing cells of the giant‐cellular xanthophycean alga Vaucheria frigida is mainly composed of cellulose microfibrils (CMFs) arranged in random directions and the major matrix component into which the CMFs are embedded throughout the cell. The mechanical properties of a cell‐wall fragment isolated from the tip‐growing region, which was inflated by artificially applied pressure, were measured after enzymatic removal of the matrix component by using a protease; the results showed that the matrix component is involved in the maintenance of cell wall strength. Since glucose and uronic acid are present in the matrix component of Vaucheria cell walls, we measured the mechanical properties of the cell wall after treatment with endo‐1,3‐ß‐glucanase and observed the fine structures of its surfaces by atomic force microscopy. The major matrix component was partially removed from the cell wall by glucanase, and the enzyme treatment significantly weakened the cell wall strength without affecting the pH dependence of cell wall extensibility. The enzymatic removal of the major matrix component by using a protease released polysaccharide containing glucose and glucuronic acid. This suggests that the major matrix component of the algal cell walls contains both proteins (or polypeptides) and polysaccharides consisting of glucose and glucuronic acid as the main constituents.     

MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及机理探究

【目的】研究MIG1基因和葡萄糖对扣囊复膜孢酵母细胞形态变化的影响及其机理探究。【方法】扣囊复膜孢酵母在不同浓度葡萄糖的YPD培养基中培养,敲除MIG1基因菌株在常规YPD培养基中培养,研究细胞内葡聚糖酶和几丁质酶活性以及细胞壁β-葡聚糖和几丁质含量与细胞形态变化之间的关系。【结果】培养基中葡萄糖浓度越低,扣囊复膜孢酵母菌丝体越少,单细胞酵母越多,且葡聚糖酶和几丁质酶活性越高,β-葡聚糖和几丁质含量越低;葡萄糖浓度对敲除MIG1基因菌株没有显著影响,葡聚糖酶和几丁质酶活性始终保持在较高水平,β-葡聚糖和几丁质含量也较低,菌体多以单细胞酵母形式存在。【结论】MIG1基因和葡萄糖通过葡萄糖阻遏作用调节葡聚糖酶和几丁质酶活性,进而影响细胞壁的葡聚糖和几丁质含量,最终影响扣囊复膜孢酵母细胞的形态变化。     

Changes in the Autolytic Activities of Maize Coleoptile Cell Walls during Coleoptile Growth

Autolytic activities of coleoptile cell walls were measuredin developing maize seedlings. The major neutral sugar componentsof the cell wall polysaccharides were arabinose, xylose andglucose. The quantities of all these components per coleoptileincreased for 5 d after germination, suggesting that levelsare augmented by biosynthetic processes during coleoptile growth.However, cell wall preparations isolated from the coleoptilesalso revealed increasing rates of autolytic activity directedtoward each of the sugar components. This result suggests thatthe constitutive hydrolytic activities expressed by cell wallsalso increase as a function of coleoptile age. The proportionof glucose in autolysis products relative to that present inthe cell walls specifically increased with coleoptile age, whilethe ratios for arabinose and xylose decreased. Kinetic analysesof autolysis demonstrated that the reactions specific for pentosesat the early growth stage are transient events and that initiallow rates of glucan autolysis increased sharply and persistedlonger. In these experiments the products of glucan autolysiswere largely monomeric while those of the pentose-specific reactionsconsisted of both monomeric and polymeric sugars. Based on theseresults, we concluded that two distinct phases of autolyticactivities are expressed in the mediation of cell wall polysaccharidemetabolism in situ. (Received July 17, 1996; Accepted November 25, 1996)     

Role of Enzymes in Growth and Morphology of Neurospora crassa: Cell-Wall-Bound Enzymes and Their Possible Role in Branching

The cell wall of Neurospora crassa contains bound enzymes that can digest its structural polymers. These enzymes are not present at the same levels at all stages of growth. The levels of these autolytic enzymes vary and generally show some relationship to the process of branching. These enzymes were removed from the cell wall by β-mercaptoethanol extraction and were tested for activity against isolated cell wall fractions. Such studies, as well as autolytic studies, showed that enzymes acting on the protein portion of the cell wall (proteases) are more prominent than enzymes that act on the glucan portion (glucanases) of the cell wall. Comparative studies between the wild type and a spreading colonial mutant spco-1 showed that earlier and higher frequency of branching in spco-1 was correlated with a greater amount of these enzymes bound to the cell walls. It is concluded from these observations that autolytic enzymes acting on the protein and glucan portion of the cell walls occur as wall-bound and participate in the process of branching in Neurospora.     

Cell wall autolytic activities and distribution of cell wall glucanases in Zea mays L. seedlings

Exo- and endo-glucanases mediate specific degradation of cell wall (1,3)(1,4)-beta-D-glucans and these enzymes have been related to auxin-mediated growth and development of cereal coleoptiles. However, their distribution and functions have not been well established in other tissues. In this study the glucanase activities and cell wall autolytic activities of different maize organs were determined. Autolysis assays serve to evaluate the hydrolysis of cell wall polymers in situ by measuring the sugars released from the insoluble cell wall matrix resulting from the action of bound enzymes. Autolytic activities were observed in the cell walls of elongating young leaves, mesocotyl and roots of maize. Wall proteins extracted from all of these structures are enriched in several types of glucanases and other wall polysaccharide hydrolases. These enzymes therefore appear to have a widespread and fundamental role in wall metabolism in growing tissues.     

Cell Wall Autolysis During Kiwifruit Development

Cell walls prepared from developing kiwifruits showed autolyticactivity. The proteins extracted from active walls were alsoable to release carbohydrates from inactive cell walls. Analysisof the sugars released, using both procedures, showed that uronicacids were the major component, especially during the firsthours of incubation, although neutral sugars such as glucoseand galactose were also present. Most of the carbohydrates autolyticallyreleased from the cell wall eluted in the void volume on a BioGel P2 column. However, carbohydrates released from inactivecell walls by the protein extract mostly eluted in the monosaccharideuronic acid and glucose peaks. The autolytic activity of isolatedcell walls, as well as the glycosylhydrolase activity of theproteins extracted from the cell walls, showed important changesduring fruit development. The differences between autolyticactivity and the glycosylhydrolase activity against the cellwall suggest that the glycosylhydrolases ‘in muro’are subjected to some regulatory mechanism which disappearswith their extraction. Finally, the role of glycosylhydrolases,such as polygalacturonases and galactosidases, in relation tocell wall changes in fruits, is discussed.Copyright 1998 Annalsof Botany Company Actinidia deliciosa; autolysis; cell wall enzymes; fruit growth; glycosylhydrolases; kiwifruit.     

Synthase III-dependent chitin is bound to different acceptors depending on location on the cell wall of budding yeast

In yeast, chitin is laid down at three locations: a ring at the mother-bud neck, the primary septum and, after cytokinesis, the cell wall of the daughter cell. Some of the chitin is free and the remainder attached to beta(1-3)glucan or beta(1-6)glucan. We recently reported that the chitin ring contributes to the prevention of growth at the mother-bud neck and hypothesized that this inhibition is achieved by a preferential binding of chitin to beta(1-3)glucan at that site. Here, we devised a novel strategy for the analysis of chitin cross-links in [14C]glucosamine-labeled cell walls, involving solubilization in water of alkali-treated walls by carboxymethylation. Intact cell walls or their digestion products with beta(1-3)glucanase or beta(1-6)glucanase were carboxymethylated and fractionated on size columns, and the percentage of chitin bound to different polysaccharides was calculated. Chitin dispersed in the wall was labeled in maturing unbudded cells and that of the ring in early budding cells. The former was mostly attached to beta(1-6)glucan and the latter to beta(1-3)glucan. This confirmed our hypothesis and indicated that the cell has mechanisms to attach chitin, a water-insoluble substance, synthesized here through chitin synthase III, to different acceptors, depending on location. In contrast, most of the chitin synthase II-dependent chitin of the primary septum was free, with the remainder linked to beta(1-3)glucan.     

Autolysis of the cell wall {beta}-D-glucan in corn coleoptiles

Corn coleoptile cell walls prepared and incubated in bufferautolyzed as much as 100 µg per mg dry weight over a 36hr period. This activity was attributed to the release of ß-D-glucanwhich constitutes as much as 110 µg per mg of the cellwall on a dry weight basis. Gel exclusion chromatography (Bio-gelP-2) of the autolytically solubilized products revealed thepresence of a polymeric component and a monosaccharide, andtime course studies showed that the polymeric component wasprogressively converted to monosaccharide. Glucose was the onlymonosaccharide detected. Treatment of the polymeric componentwith a bacterial glucanase specific for ß(13):ß(14)mixed-linkage glucans yielded distinctive tetra- and trisaccharideswhich is consistent with the hypothesis that it was derivedfrom wall ß-D-glucan. At least 90% of the autolysisproducts were derived from this wall component. The tolerance of autolytic activity to detergents and high saltconcentrations provided evidence that the enzymes responsibleare strongly associated with the wall. (Received October 3, 1978; )     

Structure of the glucan-binding sugar chain of Tip1p, a cell wall protein of Saccharomyces cerevisiae

Tip1p is one of the major cell wall mannoproteins of Saccharomyces cerevisiae and is presumed to be synthesized as a glycosylphosphatidylinositol (GPI)-anchored form. We purified Tip1p from a glucanase extract of yeast cell walls and analyzed the sugar chain involved in the cell wall linkage. One mol of glucanase-extracted Tip1p contained 7.5 mol of glucose derived from glucan and 1 mol of ethanolamine, a component of the GPI anchor. One mol of the C-terminal peptide of Tip1p digested with Achromobacter protease I also contained 7.9 mol of glucose and 1 mol of ethanolamine. On the other hand, Tip1p contained no glucosamine, which is a component of the GPI anchor. The glucan-binding sugar chain of Tip1p was released by hydrazinolysis and isolated. This sugar chain contained ethanolamine with a free amino group and a glucose reducing end, but no mannose reducing end. Phosphodiesterase treatment eliminated the free amino group from this sugar chain, suggesting that a phosphodiester bond exists between the ethanolamine and the glucan remnant. These results indicate (1) the glucan-binding sugar chain of Tip1p is a GPI derivative, and (2) the GPI anchor is cleaved at the glycosyl moiety, and the resultant mannose reducing end is probably used to link Tip1p to cell wall glucan.     

Lyticase: Endoglucanase and Protease Activities That Act Together in Yeast Cell Lysis

Yeast lytic activity was purified from the culture supernatant of Oerskovia xanthineolytica grown on minimal medium with insoluble yeast glucan as the carbon source. The lytic activity was found to consist of two synergistic enzyme activities which copurified on carboxymethyl cellulose and Sephadex G-150, but were resolved on Bio-Gel P-150. The first component was a β-1,3-glucanase with a molecular weight of 55,000. The K_m for yeast glucan was 0.4 mg/ml; that for laminarin was 5.9 mg/ml. Hydrolysis of β-1,3-glucans was endolytic, yielding a mixture of products ranging from glucose to oligomers of 10 or more. The size distribution of products was pH dependent, smaller oligomers predominating at the lower pH. The glucanase was unable to lyse yeast cells without 2-mercaptoethanol or the second lytic component, an alkaline protease. Neither of these agents had any effect on the glucanase activity on polysaccharide substrates. The protease had a molecular weight of 30,000 and hydrolyzed Azocoll and a variety of denatured proteins. The enzyme was unusual in that it had an affinity for Sephadex. Although the activity was insensitive to most protease inhibitors, it was affected by polysaccharides; yeast mannan was a potent inhibitor. The enzyme did not have any mannanase activity, however. Neither pronase nor trypsin could substitute for this protease in promoting yeast cell lysis. A partially purified fraction of the enzymes, easily obtained with a single purification step, had a high lytic specific activity and was superior to commercial preparations in regard to nuclease, protease, and chitinase contamination. Lyticase has been applied in spheroplast, membrane, and nucleic acid isolation, and has proved useful in yeast transformation procedures.     

Distribution and structure of mixed linkage glucan at different stages of elongation of maize root cells

Distribution and structure of mixed linkage glucan in the cell walls at different stages of elongation were investigated in the roots of 4-day-old seedlings of maize (Zea mays L.). Mixed linkage glucan was immunocytochemically detected already in the meristem, predominantly in the periclinal cell walls. The antibody binding by the cell walls increased in the zone of cell elongation initiation, was high during the whole process, and did not decrease in the cells whose elongation was over. The content of polysaccharide determined biochemically also rose from meristematic zone to the zone where elongation was over, amounting to 8% of dry weight and remained on the same level after the completion of cell elongation. At different stages of elongation growth, the structure of polysaccharide was not the same. In the beginning of elongation, molar ratio between trimer and tetramer (DP3/DP4) among the products of polysaccharide hydrolysis by lichenase was 3.56 ± 0.04, and after its termination it became 3.04 ± 0.09. According to literature data, such changes tell on the physical properties of polysaccharide, which along with a drastic activation of its deposition associated with the initiation of elongation make it possible to attribute the mixed linkage glucan to the factors directly affecting cell wall extensibility and therefore elongation growth.     

Autolysis of the cell wall. Its possible role in endogenous and IAA-induced growth in epicotyls of Cicer arietinum

Indoleacetic acid (IAA), a factor that induces growth in epicotyls of cicer arietinum L. cv. Castellana, increases the autolytic capacity of the cell walls by 50%, suggesting that autolysis is related to the processes of cell wall loosening that accompany growth. IAA promotes an increase in the specific activities of the enzymes involved in autolysis, mainly α-galactosidase (EC 3.2.1.22). This relationship autolysis-growth. was also observed in a study of the autolytic capacity of cell walls from regions of the epicotyl with different growth capacity. The sugars released and the level of enzymatic protein were higher in the subapical region that towards the base.     

Cell wall localization of the natural substrate of a β-galactosidase, the main enzyme responsible for the autolytic process of Cicer arietinum epicotyl cell walls

The Hw pectic fraction, extracted with hot water, is the major component of 4 days old epicotyl cell walls of Cicer arietinum L. cv. Castellana and is formed of arabinose and galactose, with smaller amounts of rhamnose, xylose, glucose and mannose. The cell wall 2βIII enzymatic fraction, with β-galactosidase activity (EC 3.2.1.23) and the main enzyme responsible for the autolytic process, essentially acts on the Hw fraction, and is able to hydrolyze 560 μg of this fraction per g of epicotyls, releasing mainly galactose as monosaccharide.
The 2βIII fraction acts very weakly on the other polysaccharide fractions of the cell wall, both pectic and hemicellulosic, releasing 80, 60 and 14 μg per g of epicotyls from the fractions extracted with oxalate (Ox), KOH 10% (KI) and KOH 24% (KII), respectively. It can be concluded that the natural substrate of this enzyme is the Hw pectic fraction, probably an arabinogalactan that is found in the cell wall in isolated form or as side chains of the rhamnogalacturonan I.     

Cell wall structure regulates the autolytic process throughout growth of Cicer arietinum epicotyls

The autolytic process in epicotyl cell walls of Cicer arietinum L. cv. Castellana, and also the hydrolysis of heat-inactivated cell walls as mediated by a cell wall β-galactosidase (EC 3.2.1.23) (named βIII and previously characterized as responsible for the autolysis), are maximal on the fourth day of germination and coincide with the maximal growth capacity. They decrease during the following days, in which the growth rate diminishes. In both cases, no differences were observed in the percentages of the different sugars released, galactose being the principal one. The βIII fraction from aged epicotyl cell walls hydrolyzed young walls in proportion to its specific activity, and more efficient than when cell walls from aged material were used as the substrate. The βIII fraction from 4 day-old epicotyls (the time for maximal autolysis) was incapable of hydrolyzing aged epicotyl cell walls to the same extent as young ones. These results, together with the levels and activity of the enzyme throughout growth, allow the assumption that the variations in the autolysis and hydrolysis caused by βIII during growth processes are due to structural modifications in the cells walls, modifications that would limit access of the enzyme to its substrate, thus impeding the release of galactose, even though the enzyme is present.