Coleoptile Cell Walls Research Articles

Effects of UV-Irradiation on Enzymic Digestion of Barley Coleoptile Cell Walls

Summary UV-irradiation caused significant changes in the digestion of cell walls from dark-grown barley ( Hordeum vulgare cv. Conquest) coleoptiles by a partially purified xylanase from culture filtrates of Aspergillus niger grown on lignocellulose. Irradiation resulted in reduced rate and extent of solubilization of pentoses and changes in their neutral sugar composition and molecular weight distribution. GC-MS analysis of irradiated cell walls indicated the presence of significant quantities of the esterified cis -isomer of ferulic acid but no measurable quantities of the cyclobutane dimers of hydroxycinnamic acids.

Changes in Esterification of the Uronic Acid Groups of Cell Wall Polysaccharides during Elongation of Maize Coleoptiles

Cell walls of grasses have two major polysaccharides that contain uronic acids, the hemicellulosic glucuronoarabinoxylans and the galactosyluronic acid-rich pectins. A technique whereby esterified uronic acid carboxyl groups are reduced selectively to yield their respective 6,6-dideuterio neutral sugars was used to determine the extent of esterification and changes in esterification of these two uronic acids during elongation of maize (Zea mays L.) coleoptiles. The glucosyluronic acids of glucuronoarabinoxylans did not appear to be esterified at any time during coleoptile elongation. The galactosyluronic acids of embryonal coleoptiles were about 65% esterified, but this proportion increased to nearly 80% during the rapid elongation phase before returning to about 60% at the end of elongation. Methyl esters accounted for about two-thirds of the total esterified galacturonic acid in cell walls of unexpanded coleoptiles. The proportion of methyl esters decreased throughout elongation and did not account for the increase in the proportion of esterified galactosyluronic acid units during growth. The results indicate that the galactosyluronic acid units of grass pectic polysaccharides may be converted to other kinds of esters or form ester-like chemical interactions during expansion of the cell wall. Accumulation of novel esters or ester-like interactions is coincident with covalent attachment of polymers containing galactosyluronic acid units to the cell wall.

Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air

Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air

Correlation between cell wall extensibility and the content of diferulic and ferulic acids in cell walls of Oryza sativa coleoptiles grown under water and in air

Rice (Oryza sativa L. cv. Sasanishiki) coleoptiles grown under water achieved greater length than those grown either in air or under water with constant air bubbling. The extensibility of cell walls in coleoptiles grown under water was larger than that in the other treatments. Per unit length of the coleoptile, the content of ferulic and diferulic acids ester‐linked to hemicelluloses was higher in air and bubbling type coleoptiles than in water type ones. The extensibility of the coleoptile cell walls correlated with the content of diferulic acids per unit length and per hemicellulose, suggesting that the enhancement of the formation of diferulic acid bridges in hemicelluloses in air or under water with air bubbling makes the cell walls mechanically rigid; thereby inhibiting cell elongation in rice coleoptiles. In addition, the ratio of diferulic acid to ferulic acid was almost constant irrespective of coleoptile age, zone and growth conditions, suggesting that the feruloylation of hemicelluloses is rate‐limiting in the formation of diferulic acid bridges in the cell walls of rice coleoptiles.

Xyloglucan Antibodies Inhibit Auxin-Induced Elongation and Cell Wall Loosening of Azuki Bean Epicotyls but Not of Oat Coleoptiles

Polyclonal antibodies were raised in rabbits against isoprimeverose (Xyl(1)Glc(1)), xyloglucan heptasaccharides (Xyl(3)Glc(4)), and octasaccharides (Gal(1)Xyl(3)Glc(4)). Antibodies specific for hepta- and octasaccharides suppressed auxin-induced elongation of epicotyl segments of azuki bean (Vigna angularis Ohwi and Ohashi cv Takara). These antibodies also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time and the relaxation rate of the cell walls) of azuki segments. However, none of the antibodies influenced auxin-induced elongation or cell wall loosening of coleoptile segments of oat (Avena sativa L. cv Victory). Auxin caused a decrease in molecular mass of xyloglucans in the cell walls of azuki epicotyls and oat coleoptiles. The antibodies inhibited such a change in molecular mass of xyloglucans in both species. Preimmune serum exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosening, or breakdown of xyloglucans. The results support the view that the breakdown of xyloglucans is associated with the cell wall loosening responsible for auxin-induced elongation in dicotyledons. The view does not appear to be applicable to poaceae, because the inhibition of xyloglucan breakdown by the antibodies did not influence auxin-induced elongation or cell wall loosening of oat coleoptiles.

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.

Selected cell wall proteins from Zea mays: Assessment of their role in wall hydrolysis

Coleoptile cell wall proteins from Zea mays L. hybrid B 37 × Mo 17 were extracted and fractionated. Three enzymes identified in that extract were examined to determine their role in cell wall hydrolysis with a goal of evaluating the extent to which they participated in autohydrolytic reactions. Two separate proteins were identified as endo‐ and exo‐glucanases. Incubation of these enzymes with heat inactivated cell walls, liberates products derived from the constitutive (1→3), (1→4)‐β‐d‐glucan. The release of sugars from walls resembles that of cell wall autolysis. A third cell wall protein degraded polysaccharides in a more general manner, releasing carbohydrates containing xylose, arabinose, galactose and glucose. Polyclonal antibodies raised against the exoglucanase protein suppressed autolytic reactions of isolated cell 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.

Enzymic Analysis of Feruloylated Arabinoxylans (Feraxan) Derived from Zea mays Cell Walls

Structural features of feruloylated arabinoxylan (feraxan) present in Zea mays L. (hybrid B 73 x Mo 17) coleoptile cell walls have been studied using a purified feraxan-dissociating enzyme (feraxanase) and an alpha-arabinofuranosidase. This experimental approach has demonstrated the following. (a) Feraxanase dissociated ca. 20% (dry weight basis) of the maize wall preparation. The predominant oligosaccharides enzymically liberated were allocated into seven major subfractions designated A-1 (0.8%), B-1 (1.6%), B-2 (2.4%), B-3 (4.6%), C-1 (1.0%), C-2 (4.2%), and C-3 (0.3%). Values in parentheses reflect the percentage of the wall associated with each subfraction. Subfractions represent samples enriched in different degrees of polymerization, sugar composition, linkage arrangements, and phenolic acid content. (b) B-1, B-2, and B-3 fractions are not feruloylated and have smaller molecular mass (less than 10(4) kilodaltons) and consist chiefly of t-arabinosyl-5-arabinosyl, 4-xylosyl, 2,4/3,4-xylosyl, and glucuronosyl residues, suggesting that these fragments constitute nonferuloylated regions of arabinoxylan. (c) C-2 and C-3 fractions contain ferulic acid (6.2% and 12.1%, respectively) and are similar to the B series in their sugar linkage arrangements but were derived from feruloylated regions. (d) Alkali treatment of the C-2 fraction decreases the molecular size of the fragment and liberates phenolic acids. The results suggest the presence of alkaline-labile links, probably diferulate bridges. (e) A-1 and C-1 fractions are larger (more than 5 x 10(5) kilodalton) and contain t-galactosyl-, 4-galactosyl, 2,4-rhamnosyl-residues, galacturonic acid, and the sugar linkage arrangements common to other fractions. The A-1 fraction is not feruloylated, whereas C-1 fraction contains 0.5% ferulic acid. The presence of galactose, rhamnose, and galacturonic acid suggests that pectic polymers, probably homopolygalacturonans and rhamnogalacturonans, are linked to nonferuloylated and feruloylated segments of arabinoxylans.

Antibodies as probes for the study of location and metabolism of (1→3), (1→4)‐β‐D‐glucans

Polyclonal antibodies, raised against ((1→3), (1→4)‐β‐D‐glucans from oat (Avena sativa L.) caryopsis, were used to investigate the location and the metabolism of mixed‐linked β‐D‐glucans. The binding of these antibodies to the cell walls of oat coleoptiles was shown by an indirect fluorescence method. Distinct fluorescent regions were observed along the inner layers of the walls of each cell. The preimmune serum or antibodies pretreated with oat caryopsis β‐D‐glucans did not react with the cell walls. Glucan antibodies were bound to the walls of other Poaceae coleoptiles as well as to those from oat mesocotyls and roots, whereas they were not bound to the walls of some dicotyledons tested. The relative glucan content of the cell walls of oat coleoptiles as determined by β‐D‐glucanase (EC 3.2.1.73) treatment was maximum between day 3 and 4 after soaking, but it declined during further elongation. A rapid decrease in glucan content was observed in excised coleoptiles when auxin or β‐D‐glucanase was present. There was a clear correlation between the glucan content expressed on a basis of cell wall polysaccharides and the amount of the antibodies bound to the cell walls. These results indicate that the antibodies are useful probes to detect and determine (1→3), (1→4)‐β‐D‐glucans of cell walls.

Immunocytochemical Localization of a Wheat Germ Lysozyme in Wheat Embryo and Coleoptile Cells and Cytochemical Study of Its Interaction with the Cell Wall

Among several wheat (Triticum aestivum L.) germ proteins able to lyse Micrococcus lysodeikticus, one lysozyme (W1A) was purified by ion-exchange chromatography, gel filtration, and preparative polyacrylamide gel electrophoresis. Polyclonal antibodies against this lysozyme were raised in rabbits. The in situ localization of W1A lysozyme was achieved by the indirect protein A-gold technique. Large amounts of W1A lysozyme were found in cell walls whereas intercellular spaces, cytoplasm, and organelles were nearly free of labeling. Specificity of labeling was assessed with several controls. In an attempt to detect the presence of binding sites, W1A lysozyme was complexed to colloidal gold. Particles were specifically distributed in large amounts over wheat embryo and coleoptile cell walls. The absence of labeling over isolated coleoptile cell walls treated with 0.1 and 0.4 molar potassium hydroxide for hemicellulose extraction indicated that W1A lysozyme binding sites were probably of hemicellulosic nature.

Changes in the hemicellulosic polysaccharides of Avena coleoptile cell walls during growth of intact seedlings

The variations taking place in the major polysaccharides of the cell wall of Avena sativa L. cv. Victory coleoptiles were studied during growth. Two fractions were obtained, one corresponding to cellulose and the other to the hemicelluloses. The relative content of cellulose was greater in the basal regions of the coleoptiles and the proportion of this polysaccharide fraction increased in all regions during growth. The hemicellulosic fraction was composed of two parts, an insoluble one designated the HC A hemicellulosic fraction, whose main component was seen to be an insoluble xyloglucan, and a soluble part designated the HC B hemicellulosic fraction, composed of an arabinoxylan and a (β1-3)- or (β1-4)-glucan. This latter was degraded during the growth of the coleoptiles. The average molecular weight of hemicelluloses HC A and HC B decreased from the sub-apical zones to the basal zones of the coleoptiles; this was related to the growth capacity of each of the regions into which the coleoptiles were divided.

Molecular cloning of cDNAs encoding a putative cell wall protein from Zea mays and immunological identification of related polypeptides

Copy DNAs corresponding to a highly repetitive, proline-rich protein from maize have been cloned by differential screening of a coleoptile cDNA library. The deduced amino acid sequence contains a single repetitive element of carrot extensin (Ser-Pro-Pro-Pro-Pro). The related mRNAs have a defined distribution in tissues of the plant and are accumulated mainly in the coleoptile node and root tip. A peptide that corresponds to one of the repetitive elements of the protein has been synthesized and antisera have been obtained in rabbits. These antibodies react against crude preparations of coleoptile cell wall and against polypeptides extracted following the protocols described for the extraction of extensin. From these data it is concluded that the cDNAs correspond to a family of cell wall glycoproteins from maize.

Inhibiting Effect of Auxin on Glucosamine Incorporation into Cell Walls of Rice Coleoptiles

Inhibiting Effect of Auxin on Glucosamine Incorporation into Cell Walls of Rice Coleoptiles

Changes in the Molecular Weight Distribution of the Hemicellulosic Polysaccharides from Rice Coleoptiles Growing Under Different Conditions

The changes in the molecular weight distribution of water-soluble and water-insoluble hemicelluloses from the cell walls of rice coleoptiles growing in air and under water were studied. The growth of rice coleoptiles was remarkably enhanced by growing under water. Water-insoluble hemicellulose, mainly constituted by xyloglucan, suffered an important depolymerization during coleoptile growth. On the other hand, β-glucan and arabinoxylan, the two main polysaccharides of the water-soluble hemicelluloses showed different changes during coleoptile growth. β-glucan showed an increase in its degree of polymerization during the coleoptile fast growth phase and it decreased before coleoptile growth ceased. Arabinoxylan did not show important differences in its mass-average molecular weight. Thus, xyloglucan and β-glucan are the two hemicellulosic polysaccharides involved in the cell wall loosening mechanism during coleoptile growth under both culture conditions.

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.

<italic>N</italic>-Acetylglucosamine-Containing Glycopeptide Released from Rice Coleoptile Cell Walls by Protease Treatment

<italic>N</italic>-Acetylglucosamine-Containing Glycopeptide Released from Rice Coleoptile Cell Walls by Protease Treatment

A gas chromatographic analysis of the release of arabinose from coleoptile cell walls under the influence of auxin and dextranase preparations

An analysis by gas chromatography of the products of digestion of cell wall pellets of Avena sativa L., coleoptiles shows that after short term autolysis of pellets from auxin-rich plants, arabinose is released. Addition of arabinan as a substrate increases the yield of arabinose considerably, suggesting that a similar substance is broken down in the walls. In pellets from auxin-poor coleoptiles much less arabinose is released and addition of arabinan has no effect. Dextranase preparations stimulate this release and can also break down arabinan. This suggests that auxin and a dextranase-like enzyme are involved in the process.

Tunicamycin‐induced growth and inhibition of glucosamine incorporation into cell walls of rice coleoptiles

Tunicamycin, 0.25 to 2.5 μM, promotes elongation of rice coleoptile sections after a 2 h lag. Tunicamycin decreased the minimum stress‐relaxation time of the cell wall, T0; and the wall loosening is recognized as the cause of this growth promotion. Bacitracin did not have significant effects on growth or T0 except for inhibition of elongation at high concentration. Coleoptile sections were incubated with [14C]‐glucosamine, and the synthetic pathway of the hexosamine‐containing cell wall component was examined by a pulse‐chase experiment. This component seems to be synthesized in the particulate fraction and secreted mainly into the hemicellulose I fraction. Tunicamycin strongly inhibited glucosamine incorporation into the particulate fraction and stopped the labeling of the cell wall. At 2.5 μM, tunicamycin had no effect on incorporation of mannose, leucine or proline. These results suggest that the hexosamine‐containing wall component is a kind of asparagine‐linked glycoprotein, and that this component plays a principal role in formation of the cell wall network and in growth regulation.

Transient Nature of a (1 → 3), (1 → 4)-β-d-Glucan in Zea mays Coleoptile Cell Walls

Excised Zea mays L. embryos were cultured on Linsmaier and Skoog medium. Coleoptiles were sampled at regular intervals and the length, fresh weight, cell wall weight, and cell wall neutral sugar composition were determined. A specific beta-d-glucanase from Bacillus subtilis was used to determine the content of a (1 --> 3),(1 --> 4)-beta-d-glucan.Coleoptiles elongated through the 5th day following imbibition with the most rapid elongation occurring between days 3 and 4. The greatest net rate of incorporation of cell wall per coleoptile occurred between the 2nd and 3rd days when deposition of approximately one-third of the maximum net glucan level was observed. By day 5, the amount of glucan present had increased 34-fold from the 6 micrograms per coleoptile on day 1 and accounted for about 14% of the cell wall (w/w). Thereafter, the glucan content declined until only 3.3% (w/w) remained by day 10. In this 10-day interval, xylose increased 32% and cellulose content doubled, while proportions of other neutral sugars changed less dramatically.These results are consistent with a possible role for the beta-d-glucan in elongation of the Zea coleoptile. Moreover, changes in the quantity of this wall component clearly reflect the dynamic nature of plant cell wall polysaccharides. An evaluation of glucan dynamics in vivo suggests that in vitro autolysis studies employing Zea coleoptile walls may overestimate the actual rate of glucan turnover in the intact tissue.