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.     

Auxin-enhanced glucan autohydrolysis in maize coleoptile cell walls

Cell walls isolated from auxin-pretreated maize (Zea mays L.) coleoptile segments were assayed to disclose evidence for the existence of enhanced autolysis. To improve the sensitivity of the measurements and to facilitate kinetic analysis, isolated cell walls were consolidated within a small column, and the autolysis rate was directly determined from the sugar content of the effluent. This protocol revealed that the maximum rate of autohydrolysis of walls prepared from segments occurs within the first 2 hours and a steady decline commences almost immediately. Walls from indoleacetic acid pretreated segments (0.5-4 hours) released sugar at a higher rate initially (110-125% of controls) and the enhanced rate of autolysis continued for 6 to 8 hours, but then it became equivalent to that of the controls. Pretreatment of the segments at acidic pH had no effect on the measurable rates of autolysis. The (1→3), (1→4)-β-d-glucan content of the walls and the extractable glucanase activities support the hypothesis that temporal enhancement of autohydrolysis is a function of auxin on enzyme activity. The progressive decline in autolysis during prolonged incubations is consistent with the decrease in the quantity of the β-d-glucan in the wall. The relationship between glucan content and autolysis rate is supported by the observation that while glucose pretreatment of segments had only a small effect on initial autolysis rates, the presence of the sugar during pretreatment served to extend the interval over which higher rates of autolysis could be sustained. The results demonstrate that autolysis is related to auxin-induced wall metabolism in maize coleoptiles.     

EFFECT OF AUXIN ON β-1, 3-GLUCANASE ACTIVITY IN AVENA COLEOPTILE

When the homogenate of Avena coleoptile segments was fractionated, the specific activity of β-1, 3-glucanase was remarkably associated with the cell wall, partly to be released from it by a detergent. The cell wall-bound glucanase activity was increased by the treatment of coleoptile segments with auxin. Only in 10 min of the treatment the glucanase activity and the incorporation of labeled leucine into the proteins were found to be increased in the fraction to be liberated by detergent from the cell wall fraction. These effects of auxin were inhibited by 10 μg/ml cycloheximide.     

Cell elongation and metabolic turnover of the cell wall as affected by auxin and cell wall degrading enzymes

A cell wall fraction (pectic substances) of oat coleoptile segmentsfed with ¹⁴C-glucose contained more radioactivity under theeffect of auxin than did the control. When labeled segmentswere grown for 6 hr in auxin or glucanase solution the labelin the hemicellulose fraction decreased as growth increased.ß-1,3-Glucanase prepared from the culture of a fungus,Sclerotinia libertiana, induces elongation of segments of thepea stem and the oat coleoptile. Traces of cellulase and pectinmethylesterase contaminating the enzyme preparation are notresponsible for the stimulatory effect. Cellulase seemed tobe rather inhibitory and pectin methylesterase showed only aslight effect on coleoptile elongation. A possible relationshipbetween the metabolic turnover of hemicellulosic polysaccharideand cell wall extension is suggested. (Received February 5, 1968; )     

Auxin- and hydrogen ion-induced cell wall loosening and cell extension in Avena coleoptile segments

Hydrogen ions and auxin induce rapid cell extension of Avenacoleoptile segments. Nojirimycin (5-amino-5-deoxy-D-glucopyranose),a potent glucanase inhibitor, inhibits auxin-induced growthbut does not affect hydrogen ion-induced extension. This inhibitorhas little effect on respiration of coleoptile segments butstrongly inhibits the in vitro activity of ß-glucosidase.Hydrogen ions and auxin decreased the minimum stress-relaxationtime of the cell wall, indicating that both enhanced cell extensionthrough cell wall loosening. The hemicellulosic glucose contentof the cell wall which was decreased by auxin after about a2-hr lag, was not affected by hydrogen ions. These results suggestthat cell wall loosening induced by hydrogen ions may not bethe same as that caused by auxin, although both phenomena arerepresented by the decrease in the minimum stress-relaxationtime. (Received November 1, 1976; )     

Galactose inhibition of auxin-induced cell elongation in oat coleoptile segments

Auxin-induced cell elongation in oat coleoptile segments was inhibited by galactose; removal of galactose restored growth. Galactose did not appear to affect the following factors which modify cell elongation: auxin uptake, auxin metabolism, osmotic concentration of cell sap, uptake of tritium-labeled water, auxin-induced wall loosening as measured by a decrease in the minimum stress-relaxation time and auxininduced glucan degradation. Galactose markedly prevented incorporation of [¹⁴C]-glucose into cellulosic and non-cellulosic fractions of the cell wall. It was concluded that galactose inhibited auxin-induced long-term elongation of oat coleoptile segments by interfering with cell wall synthesis.     

Preparation and Properties of a beta-d-Glucanase for the Specific Hydrolysis of beta-d-Glucans

A beta-d-glucanase highly specific for glucans containing a linkage sequence ... Glc 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc ... has been isolated from several commercial preparations of Bacillus subtilis alpha-amylase including one purified by repeated crystallization. The beta-d-glucanase will not hydrolyze cellulose or laminarin. Gel filtration on a Bio-Gel P-200 column results in separation of the glucanase from the alpha-amylase. The enzyme is of the endo type as changes in the substrate viscosity appear long before the appearance of detectable reducing sugars. No evidence of product inhibition was revealed and appropriate substrates were converted to oligosaccharides, the quantity of which approaches theoretical yields. The products of the reaction were separated according to molecular size by use of Bio-Gel P-2 gel filtration and found to be consistent with the action pattern of the enzyme. Kinetic studies show that the enzyme has an optimum activity at pH 6.5, a V(max) of 13.9 mug glucose equivalent released/mug protein.hour, and an apparent Km of 3.4 mg of lichenan per ml. Potential application of this enzyme for the structural characterization of plant cell wall glucans is discussed.     

Role of Expansin in Cell Enlargement of Oat Coleoptiles (Analysis of Developmental Gradients and Photocontrol)

Expansins are wall proteins that mediate a type of acid-induced extension in isolated plant cell walls (S. McQueen-Mason, D.M. Durachko, D.J. Cosgrove [1992] Plant Cell 4: 1425-1433). To assess the role of these proteins in the process of cell enlargement in living tissues, we compared the spatial and temporal growth patterns of oat (Avena sativa L.) coleoptiles with four wall properties related to expansin action. These properties were (a) the ability of isolated walls and living segments to extend in acidic buffer, (b) the ability of heat-inactivated walls to extend upon application of expansins, (c) the amount of immunologically detectable expansin in wall protein extracts, and (d) the extractable expansin activity of walls. Growth rate was maximal in the apical half of dark-grown coleoptiles and negligible in the basal region. This growth pattern correlated with properties a and b; in contrast, the amount and activity of extractable expansin (properties c and d) were reduced only in the most basal region. Upon exposure to white light, coleoptiles abruptly ceased elongation at 8 to 10 h after start of irradiation, and this cessation correlated with reductions in properties a to c. The growth cessation at 8 to 10 h also coincided with the loss of growth response to exogenous auxin and fusicoccin in excised coleoptile segments. These results lend correlative support to the hypothesis that expansin action is important for growth responses of living oat coleoptiles (e.g. responses to acidic buffers, auxin, fusicoccin, aging, and light). Our results suggest that changes in the susceptibility of the wall to expansin action, rather than changes in expansin activity, may be a key determinant of the growth patterns in oat coleoptiles.     

Changes in the Activities and Polypeptide Levels of Exo- and Endoglucanases in Cell Walls during Developmental Growth of Zea mays Coleoptiles

Exo- and endoglucanases present in cereal coleoptile cell wallsare capable of mediating hydrolysis of non-cellulosic rß-(l,3)(l,4)-glucanin situ. To assess the relationship with cell elongation, glucanaseactivities and the respective polypeptide abundance were determinedas a function of Zea mays coleoptile development. Both exo-and endoglucanase activities were quite low initially, but increasedto achieve maximum levels by days 5 or 6. Western blots revealedthat the density of the protein bands increased with coleoptiledevelopment generally in correspondence to activity levels.However, in bioassays with 3 d old coleoptile segments we foundthat auxin stimulation of glucanase activities did not resultfrom increased glucanase polypeptide levels. Hence, there wasno evidence for de novo protein synthesis in excised coleoptilesin response to added auxin. While glucanase antibodies stronglyinhibited IAA-induced elongation of coleoptile segments on days2–4, these same antibodies had little effect on day 1.We conclude that glucanases contribute to auxin mediated coleoptilegrowth only during a limited developmental interval. We proposethat when elongation is dominate, the physical properties ofthe cell wall adjust in response to metabolism of cell wallrß-(l,3)(l,4)-glucans but the enhancement of suchactivity is governed by factors other than glucanase proteinlevels. (Received December 24, 1997; Accepted April 30, 1998)     

Autolytic activities of the cell wall in rice coleoptiles. Effects of nojirimycin

Oryza sativa L. var. bahia coleoptile cell walls show sufficient autolytic activity for the release into the surrounding medium of amounts up to 60 μg of sugars per mg of dry weight of cell wall. The products released elute in Bio-gel P.2 as mono- and polysaccharides with glucose as the sole component. The polysaccharide component releases tri- and tetrasaccharides on treatment with a glucanase specific for β (1–3) (1–4) linkages in the same proportion as that of the mixed glucan of the cell wall. This supports the hypothesis that the polysaccharide component originates from the cell wall glucan and that autolysis is therefore related to the processes of the loss of rigidity of the cell wall. Nojirimycin (a specific glucanase inhibitor and inhibitor of auxin-induced elongation) decreases autolytic activity of the cell walls, reducing it to 30% of its normal value. Bio-gel P. 2 elution of the products released in autolysis in the presence of nojirimycin shows that only the monosaccharide fraction was affected.     

The hydrolytic and transferase action of alternanase on oligosaccharides.

Alternanase is an enzyme which endo-hydrolytically cleaves the alpha-(1-->3), alpha-(1-->6)-linked D-glucan, alternan. The main products are isomaltose, alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc and the cyclic tetrasaccharide cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. It is also capable of acting on oligosaccharide substrates. The cyclic tetrasaccharide is slowly hydrolyzed to isomaltose. Panose and the trisaccharide alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-D-Glc both undergo transglycosylation reactions to give rise to the cyclic tetrasaccharide plus D-glucose, with panose being converted at a much faster rate. The tetrasaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc is hydrolyzed to D-glucose plus the trisaccharide alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-D-Glc. Alternanase does not act on isomaltotriose, theanderose (6(Glc)-O-alpha-D-Glcp sucrose), or alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-alpha-D-Glc. The enzyme releases 4-nitrophenol from 4-nitrophenyl alpha-isomaltoside, but not from 4-nitrophenyl alpha-D-glucopyranoside, 4-nitrophenyl alpha-isomaltotrioside, or 4-nitrophenyl alpha-isomaltotetraoside.     

Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells

Growth of Micromonospora chalcea on a defined medium containing laminarin as the sole carbon source induced the production of an extracellular enzyme system capable of lysing cells of various yeast species. Production of the lytic enzyme system was repressed by glucose. Incubation of sensitive cells with the active component enzymes of the lytic system produced protoplasts in high yield. Analysis of the enzyme composition indicated that beta(1-->3) glucanase and protease were the most prominent hydrolytic activities present in the culture fluids. The system also displayed weak chitinase and beta(1-->6) glucanase activities whilst devoid of mannanase activity. Our observations suggest that the glucan supporting the cell wall framework of susceptible yeast cells is not directly accessible to the purified endo-beta(1-->3) glucanase and that external proteinaceous components prevent breakdown of this polymer in whole cells. We propose that protease acts in synergy with beta(1-->3) glucanase and that the primary action of the former on surface components allows subsequent solubilization of inner glucan leading to lysis.     

Effect of cycloheximide and cordycepin on auxin-induced elongation and {beta}-glucan degradation of noncellulosic polysaccharides of Avena coleoptile cell wall

The effect of cycloheximide (10^–5 M) and cordycepin (10^–4M) used as protein and RNA synthesis inhibitors, respectively,on auxin action in noncellulosic ß-glucan degradationof Avena coleoptile cell wall was investigated. Both depressedauxin-induced ßglucan degradation of the cell wallas well as auxin-induced elongation and cell wall loosening,suggesting that the process of ß-glucan degradationof the cell wall is closely associated with cell wall looseningand that auxin enhances the activity of an enzyme for ß-glucandegradation through de novo synthesis of RNA and protein butnot through activation of the enzyme in situ. Kinetic studywith the inhibitors showed that RNA metabolism involved in ß-glucandegradation was stimulated by auxin treatment of only 15 minwhile a longer lag phase (about 1 hr) existed for the synthesisof the enzyme. (Received December 16, 1978; )     

Cell wall development in maize coleoptiles

The physical bases for enhancement of growth rates induced by auxin involve changes in cell wall structure. Changes in the chemical composition of the primary walls during maize (Zea mays L. cv WF9 × Bear 38) coleoptile development were examined to provide a framework to study the nature of auxin action. This report documents that the primary walls of maize cells vary markedly depending on developmental state; polymers synthesized and deposited in the primary wall during cell division are substantially different from those formed during cell elongation.

The embryonal coleoptile wall is comprised of mostly glucuronoarabinoxylan (GAX), xyloglucan, and polymers enriched in 5-arabinosyl linkages. During development, both GAX and xyloglucan are synthesized, but the 5-arabinosyls are not. Rapid coleoptile elongation is accompanied by synthesis of a mixed-linked glucan that is nearly absent from the embryonal wall. A GAX highly substituted with mostly terminal arabinofuranosyl units is also synthesized during elongation and, based on pulse-chase studies, exhibits turnover possibly to xylans with less substitution via loss of the arabinosyl and glucuronosyl linkages.

     

Distribution of noncellulosic β-d-glucans in grasses and other monocots

Cell wall specimens from nonendospermic tissues of six grasses and representatives of nine other monocot families were treated with a specific glucanase in order to liberate wall-bound noncellulosic β-d-glucans. Gel filtration chromatography profiles of the oligosaccharides released from all grass species indicated the presence of a mixed linkage β-(1→3):(1→4)-glucan. The results also indicate that this glucan was not present in the other monocots examined. The evidence from this and previous studies indicates that the mixed linkage glucan may be restricted in the monocots to the Gramineae.     

Jasmonic Acid Inhibits the IAA-Induced Elongation of Oat Coleoptile Segments: a Possible Mechanism Involving the Metabolism of Cell Wall Polysaccharides

The effects of jasmonic acid (JA) on the IAA-induced elongationof segments of etiolated oat (Avena sativa L. cv. Victory) coleoptileswere studied. Exogenously applied JA substantially inhibitedIAA-induced elongation of oat coleoptile segments. The inhibitionof the growth of oat coleoptile segments due to JA appeared2 h after the application of JA with IAA. JA did not affectthe consumption of oxygen by the segments, the osmolarity ofthe cell sap or the IAA-induced loosening of cell walls, whichwas recognized as a decrease in the minimum stress-relaxationtime (T₀). JA was extremely effective in preventing increasesin the amount of the cell wall polysaccharides in both the non-cellulosicfraction and the cellulosic fraction during coleoptile growthin the presence and in the absence of IAA. Inhibition of thegrowth of oat coleoptile segments induced by JA was partiallyreversed by the simultaneous addition of sucrose to the testsolution. From these results, it appears that JA inhibits IAA-inducedelongation of oat coleoptile segments by interfering with someaspects of sugar metabolism that are related to the degradationand/or the synthesis of cell wall polysaccharides. (Received March 15, 1994; Accepted August 2, 1994)     

Growth capacity in response to auxin of coleoptile segments of normal and dwarf rice strains

Auxin-induced growth, epidermal cell length, cellular osmotic potential, and cell wall composition of coleoptile segments excised from one normal and two dwarf rice strains were studied 2, 3, 4, and 5 days after soaking. The auxin-induced growth was higher at the early stages of coleoptile growth and decreased with age, being always higher in normal than in the two dwarf strains. A good correlation between auxin-induced growth and auxin-induced decrease in the minimum stress-relaxation time has been found, suggesting that the different growth capacity in response to auxin among the three different strains is due to differences in the structure of their cell walls. In fact, cell wall analysis revealed that (1) the relative α-cellulose content of the cell walls was higher in the two dwarf strains than in the normal one, and (2) the auxin-induced decrease in noncellulosic glucose was high, compared with dwarf strains, in the normal strain, which showed the higher auxin-induced growth, showing a highly significant correlation between the decrease in noncellulosic glucose and the growth in response to auxin. Thus, the different growth between normal and dwarf strains might be attributed to their different capacity to degrade β-glucan of their cell walls.     

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.     

Diferulic and ferulic acid in the cell wall of Avena coleoptiles—Their relationships to mechanical properties of the cell wall

Alkaline hydrolysis liberated ferulic and diferulic acid from polysaccharides of the Avena coleoptile ( Avena sativa L. cv. Victory I) cell walls. The amount of the two phenolic acids bound to cell walls increased substantially at day 4–5 after sowing, when the growth rate of the coleoptile started to decrease. The level of these acids was almost constant from the tip to base in 3-day-old coleoptiles, but increased toward the basal zone in 4- and 5-day-old ones. The ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age and zone. An increase in the amount of ferulic and diferulic acids bound to cell wall polysaccharides correlated with a decrease in extensibility and with an increase in minimum stress-relaxation time and relaxation rate of the cell wall. The level of lignin in the cellulose fraction increased as coleoptiles aged, but this increase did not correlate with changes in mechanical properties of the cell walls. These results suggest that ferulic acid, ester-linked to cell wall polysaccharides, is oxidized to give diferulic acid, which makes the cell wall mechanically rigid by cross-linking matrix polysaccharides and results in limited cell extension growth. In addition, it is probable that the step of feruloylation of cell wall polysaccharides is rate-limiting in the formation of in-termolecular bridges by diferulic acid in Avena coleoptile cell walls.