Maize Coleoptile Cell Walls Research Articles

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

Glucanase-mediated degradation of β-(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 β-(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.

A Novel β-Glucosidase from the Cell Wall of Maize ( Zea mays L.): Rapid Purification and Partial Characterization

Plants have a variety of glycosidic conjugates of hormones, defense compounds, and other molecules that are hydrolyzed by beta-glucosidases (beta-D-glucoside glucohydrolases, E.C. 3.2.1.21). Workers have reported several beta-glucosidases from maize (Zea mays L.; Poaceae), but have localized them mostly by indirect means. We have purified and partly characterized a 58-Ku beta-glucosidase from maize, which we conclude from a partial sequence analysis, from kinetic data, and from its localization is not identical to any of those already reported. A monoclonal antibody, mWP 19, binds this enzyme, and localizes it in the cell walls of maize coleoptiles. An earlier report showed that mWP19 inhibits peroxidase activity in crude cell wall extracts and can immunoprecipitate peroxidase activity from these extracts, yet purified preparations of the 58 Ku protein had little or no peroxidase activity. The level of sequence similarity between beta-glucosidases and peroxidases makes it unlikely that these enzymes share epitopes in common. Contrary to a previous conclusion, these results suggest that the enzyme recognized by mWP19 is not a peroxidase, but there is a wall peroxidase closely associated with the 58 Ku beta-glucosidase in crude preparations. Other workers also have co-purified distinct proteins with beta-glucosidases. We found no significant charge in the level of immunodetectable beta-glucosidase in mesocotyls or coleoptiles that precedes the red light-induced changes in the growth rate of these tissues.

Polypeptide Characteristics and Immunological Properties of Exo- and Endoglucanases Purified from Maize Coleoptile Cell Walls

Summary Cereal coleoptile cell walls have exo- and endoglucanases capable of mediating the hydrolysis of non-cellulosic β-(l,3)(l,4)-glucan in situ . A purified exoglucanase (EC 3.2.1.58) resolved as a single band at 73.5 kDa, while endoglucanase isozymes consistently appeared as two bands at 32.9 and 34.3 kDa when subjected to SDS-PAGE. HPLC analysis of the native proteins by gel-permeation chromatography revealed molecular weights of ca. 55 and 29 kDa for the exo- and endoglucanases, respectively. The exoglucanase has an isoelectric focusing point at pi 7.2 and the endoglucanase isozymes appeared as two major bands, one at pi 7.8 and another at 7.3. Deglycosylation of the native proteins followed by SDS-PAGE demonstrated that sugars accounted for ca. 6.5 % of the exoglucanase and were 12.5 and 8.8 % of the two endoglucanase isozymes, respectively. After deglycosylation the two endoglucanases converged at 30.0 kDa, suggesting polypeptide homology and that divergence in electrophoretic mobility was a consequence of glycosylation. Antibodies raised against intact exo- and endoglucanases recognized the polypeptide of the corresponding enzymes, irrespective of glycosylation. The N-terminal amino acid sequence supported the conclusion that the exo- and endoglucanase have different polypeptide structures.

Autolysis Promotes the Extension Capacity of Zea mays Coleoptile Cell Walls in Response to Acid pH Solutions

The relationship between autolytic degradation of β(1–3),(1–4)-D-glucan and acid pH-induced extension of isolated Zea mays cell walls has been investigated using a constant-load extension technique. Acidic buffer (4.5) was able to induce an additional extension (Ea) on cell walls already extended at pH 6.8 buffer under a 20 g-mass load, indicating that the additional extension (Ea) was the parameter that better represented the effect of the different treatments on the mechanical properties of maize coleoptile cell walls. The additional extension in response to acidic pH was higher when cell walls had been previously autolysed for 24 h at pH 5.5. Furthermore, the acid-pH effect was dependent on the presence during the constant load extension of some thermo-labile factors, suggesting the participation of expansins. Acid pH increased Ea of native cell walls through an increase in the plastic extension (Ep) in agreement with a one step mechanism leading directly to irreversible (plastic) wall extension as suggested by Cosgrove (1977). The autolytic degradation of β(1–3), (1–4)-D-glucan was also able to modify the mechanical properties of maize coleoptile cell walls increasing its elastic extension (Ee) in response to pH 4.5 buffer but that modification only leads to an increase in wall extension when expansins are active, suggesting a cooperation between β-glucan turnover and expansin action.

Sequential extraction of dehydrodiferulates shows heterogeneity in their degree of association withZea mays coleoptile cell walls

Sequential extraction of Zea mays coleoptile cell walls with increasing concentration of potassium hydroxide (1 mM–4 M) rendered different cell wall fractions soluble: carbohydrate and phenolic analyses of each extracted fraction were performed. Ferulic acid was the main monomeric phenolic acid extracted from cell walls, while some p-coumaric and 8-8′, 8-5′, 8-O-4′ and 5-5′ dehydrodiferulic acids were also extracted, the 8-8′ dimer being the main one extracted by saponification from maize coleoptile cell walls. The different potassium hydroxide concentrations at which the various phenolic acids, both monomeric and dimeric, were extracted showed that they were associated with cell walls at different degrees. © 1998 John Wiley & Sons, Ltd.

White light promotes the formation of diferulic acid in maize coleoptile cell walls by enhancing PAL activity

To elucidate the mechanism by which white fluorescent light (5 W m‐2) stimulates the formation of diferulic acid (DFA) in cell walls, the effect of light on phenylalanine‐and tyrosine‐ammonia‐lyase (PAL, EC 4.3.1.5 and TAL, EC 4.3.1.5) and peroxidase activities was studied using coleoptiles of maize (Zea mays L. cv. Cross Bantam T51). Growth rate of dark‐grown coleoptiles was highest at the basal zone and decreased towards the tip, while continuous irradiation caused an inhibition of growth, especially at the basal zone. Light decreased the cell wall extensibility in all zones of the coleoptile. The amounts of DFA, ferulic acid (FA) and p‐coumaric acid (p‐CA) increased by severalfold in cell walls of light‐grown maize coleoptiles as compared with those grown in the dark. Strong correlations were observed between the increase in the contents of either DFA, FA or p‐CA and the decrease in cell wall extensibility. Light decreased the wall‐bound peroxidase activity. No correlation was found between DFA content and peroxidase activity. The activities of PAL and TAL were enhanced upon white light irradiation. The increment in either DFA, FA or p‐CA content was correlated with an increase in PAL activity, but not with that in TAL activity. White light may promote DFA formation in the cell walls of maize coleoptiles by enhancing PAL activity.

Structure and distribution of lignin in primary and secondary cell walls of maize coleoptiles analyzed by chemical and immunological probes

Lignin is an integral constituent of the primary cell walls of the dark-grown maize (Zea mays L.) coleoptile, a juvenile organ that is still in the developmental state of rapid cell extension. Coleoptile lignin was characterized by (i) conversion to lignothiolglycolate derivative, (ii) isolation of polymeric fragments after alkaline hydrolysis, (iii) reactivity to antibodies against dehydrogenative polymers prepared from monolignols, and (iv) identification of thioacidolysis products typical of lignins. Substantial amounts of lignin could be solubilized from the coleoptile cell walls by mild alkali treatments. Thioacidolysis analyses of cell walls from coleoptiles and various mesocotyl tissues demonstrated the presence of guaiacyl-, syringyl- and (traces of)p-hydroxyphenyl units besidesp-coumaric and ferulic acids. There are tissue-specific differences in amount and composition of lignins from different parts of the maize seedling. Electron-microscopic immunogold labeling of epitopes recognized by a specific anti-guaiacyl/syringyl antibody demonstrated the presence of lignin in all cell walls of the 4-d-old coleoptile. The primary walls of parenchyma and epidermis were more weakly labeled than the secondary wall thickenings of tracheary elements. No label was found in middle lamellae and cell corners. Lignin epitopes appeared first in the tracheary elements on day 2 and in the parenchyma on day 3 after sowing. Incubation of coleoptile segments in H2O2 increased the amount of extractable lignin and the abundance of lignin epitopes in the parenchyma cell walls. Lignin deposition was temporally and spatially correlated with the appearance of epitopes for prolinerich proteins, but not for hydroxyproline-rich proteins, in the cell walls. The lignin content of coleoptiles was increased by irradiating the seedlings with white or farred light, correlated with the inhibition of elongation growth, while growth promotion by auxin had no effect. It is concluded that wall stiffness, and thus extension growth, of the coleoptile can be controlled by lignification of the primary cell walls. Primary-wall lignin may represent part of an extended polysaccharide-polyphenol network that limits the extensibility of the cell walls.

Viscoelastic versus plastic cell wall extensibility in growing seedling organs: a contribution to avoid some misconceptions

In a recent publication (Kutschera, 1996), it was reported that the cell walls of growing rye coleoptiles exhibit irreversible (plastic) extensibility in a rheological extension test. Basically similar measurements with cell walls of maize coleoptiles had previously shown that the apparent plastic extensibility determined in this material is in reality due to the slowly reversible (viscoelastic) extensibility of the walls. A recent reinvestigation of this discrepancy showed that rye coleoptile walls also behave as a perfectly viscoelastic material if precautions are taken to prevent measuring artefacts. Similar results were obtained with cell walls from the growing zone of various other seedling organs (maize mesocotyl, maize root, cucumber hypocotyl). It is concluded that plastic extensibility has not yet been convincingly demonstrated by rheological tests that determine the intrinsic material properties of cell walls. Reported changes in mechanical material properties of cell walls produced by growth-controlling factors such as auxin or light may generally be attributed to changes in viscoelasticity which are not directly related to the chemo-rheological processes controlling wall extension of growing cells.

White light promotes the formation of diferulic acid in maize coleoptile cell walls by enhancing PAL activity

White light promotes the formation of diferulic acid in maize coleoptile cell walls by enhancing PAL activity

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

Autolytic activities of coleoptile cell walls were measured in developing maize seedlings. The major neutral sugar components of the cell wall polysaccharides were arabinose, xylose and glucose. The quantities of all these components per coleoptile increased for 5 d after germination, suggesting that levels are augmented by biosynthetic processes during coleoptile growth. However, cell wall preparations isolated from the coleoptiles also revealed increasing rates of autolytic activity directed toward each of the sugar components. This result suggests that the constitutive hydrolytic activities expressed by cell walls also increase as a function of coleoptile age. The proportion of glucose in autolysis products relative to that present in the cell walls specifically increased with coleoptile age, while the ratios for arabinose and xylose decreased. Kinetic analyses of autolysis demonstrated that the reactions specific for pentoses at the early growth stage are transient events and that initial low rates of glucan autolysis increased sharply and persisted longer. In these experiments the products of glucan autolysis were largely monomeric while those of the pentose-specific reactions consisted of both monomeric and polymeric sugars. Based on these results, we concluded that two distinct phases of autolytic activities are expressed in the mediation of cell wall polysaccharide metabolism in situ.

Distribution of pectins in cell walls of maize coleoptiles and evidence against their involvement in auxin-induced extension growth

Aiming to elucidate the possible involvement of pectins in auxin-mediated elongation growth the distribution of pectins in cell walls of maize coleoptiles was investigated. Antibodies against defined epitopes of pectin were used: JIM 5 recognizing pectin with a low degree of esterification, JIM 7 recognizing highly esterified pectin and 2F4 recognizing a pectin epitope induced by Ca2+. JIM 5 weakly labeled the outer third of the outer epidermal wall and the center of filled cell corners in the parenchyma. A similar labeling pattern was obtained with 2F4. In contrast, JIM 7 densely labeled the whole outer epidermal wall except the innermost layer, the middle lamellae, and the inner edges of open cell corners in the parenchyma. Enzymatic de-esterification with pectin methylesterase increased the labeling by JIM 5 and 2F4 substantially. A further increase of the labeling density by JIM 5 and 2F4 and an extension of the labeling over the whole outer epidermal wall could be observed after chemical de-esterification with alkali. This indicates that both methyl- and other esters exist in maize outer epidermal walls. Thus, in the growth-controlling outer epidermal wall a clear zonation of pectin fractions was observed: the outermost layer (about one third to one half of wall thickness) contains unesterified pectin epitopes, presumably cross-linked by Ca2+ extract. Tracer experiments with3H-myo-inositol showed rapid accumulation of tracer in all extractable pectin fractions and in a fraction tightly bound to the cell wall. A stimulatory effect of IAA on tracer incorporation could not be detected in any fraction. Summarizing the data a model of the pectin distribution in the cell walls of maize coleoptiles was developed and its implications for the mechanism of auxin-induced wall loosening are discussed.

Physical extensibility of maize coleoptile cell walls: apparent plastic extensibility is due to elastic hysteresis

Segments of maize (Zea mays L.) coleoptiles demonstrate plastic cell-wall extensibility (Epl) as operationally defined by the amount of irreversible strain elicited by stretching living or frozen-thawed tissue under constant load in an extensiometer (creep test). Changes of Epl are correlated with auxin- and abscisic-acid-dependent growth responses and have therefore been causally related to hormone-controlled cell-wall loosening. Auxin induces an increase of Epl specifically in the outer epidermal wall of maize coleoptiles which is considered as the growth-limiting wall of the organ. However, detailed kinetic measurements of load-induced extension of frozen-thawed coleoptile segments necessitates a revision of the view that Epl represents a true plastic (irreversible) wall deformation. Segments demonstrate no significant irreversible extension when completely unloaded between loading cycles. Moreover, Epl can be demonstrated repeatedly if the same segment is subjected to repeated loading cycles in the extensiometer. It is shown that these phenomena result from the hysteresis behaviour of the cell wall. Stress-strain curves for loading and unloading form a closed hysteresis loop, the width of which represents Epl at a particular load. Auxin-treatment of segments leads to a deformation of the hysteresis loop, thereby giving rise to an increase of Epl. These results show that the creep test estimates the viscoelastic (retarded elastic) properties rather than the plastic properties of the wall.

Effect of anti‐wall protein antibodies on auxin‐induced elongation, cell wall loosening, and β‐D‐glucan degradation in maize coleoptile segments

Antiserum raised against the LiCl extract of maize shoot cell walls suppresses auxin‐induced elongation of maize coleoptile segments. A series of polyclonal antibodies were raised against protein fractions separated from the LiCl extract of maize (Zea mays L. cv. B73 x Mo17) coleoptiles by SP‐Sephadex and Bio‐Gel P‐150 chromatography. To understand the role of cell wall proteins in growth regulation, the effect of these antibodies on auxin‐induced elongation and changes in the cell walls of maize coleoptiles was examined. Four of the fractions prepared reacted with the antiserum raised against the total LiCl extract and effectively suppressed its growth‐inhibiting activity. Only these fractions contained the proteins responsible for eliciting growthinhibiting antibodies. The antibodies capable of growth inhibition of auxin‐induced elongation of segments also inhibited auxin‐induced cell wall loosening (decrease in the minimum stress‐relaxation time of the cell walls) of segments. The antibodies raised against one of the protein fractions separated by SP‐Sephadex inhibited the autolytic reactions of isolated cell walls and the auxin‐induced decrease in (1→3), (1→4)‐β‐D‐glucans in the cell walls. Thus, the degradation of β‐D‐glucans by cell wall enzymes may be associated with the cell wall loosening that is responsible for cell elongation. Because the other antibodies did not influence the auxin‐induced degradation of (1→3), (1→4)‐β‐D‐glucanses, β‐D‐glucanases and other cell wall enzymes may cooperate in regulation of cell elongation in maize coleoptiles.

Incorporation of Proline and Aromatic Amino Acids into Cell Walls of Maize Coleoptiles

Sections excised from maize coleoptiles incorporated radioactivity from proline, tyrosine, and phenylalanine into structural components of the cell wall. Only about 2% of radioactivity from proline taken up by sections was incorporated into cell wall; about 24% of that incorporated was in hydroxyproline and the rest remained in proline. In contrast, as much as 40% of the radioactivity from phenylalanine and 30% from tyrosine was incorporated into cell wall material. Most of this radioactivity was in saponifiable ferulic acid. Small amounts of p-coumaric and diferulic acid were found, but only a small fraction of the hemicellulose can possibly be immobilized directly through cross-linking of diferulic esters. Substantial amounts of radioactivity from aromatic amino acids remained insoluble after strong alkali extractions of wall material, and a large fraction of polysaccharide was solubilized by dilute alkali following oxidation of phenolics by acidic NaClO(2). Hence, hemicellulosic material in the cell walls of maize coleoptiles may be organized and cross-linked primarily through alkali-resistant etherified aromatics.

A Partial Chemical Characterization of Maize Coleoptile Cell Walls prepared with the Aid of a Continually Renewable Filter

THE present work was undertaken with the objective (a) of preparing plant cell-wall fragments free of contamination by intact cells, cytoplasm, plastids and other cell inclusions, but containing most of the constituents of the cell wall, including proteins, and (b) of characterizing such a cell-wall fraction chemically. The procedure differed from those previously reported in that a filtration technique was employed for purification of the cell-wall fragments and in that isolation was accomplished in an essentially non-aqueous media. It is believed that the filtration technique may prove generally useful in the preparation of cellular components where purification by differential or gradient centrifugation proves impossible. Previous reports of the chemical composition of plant cell-wall tissue have been based on the residue remaining after extraction of tissue macerates with water1. Such residues undoubtedly contain particulate cellular inclusions.