Coleoptile Cell Walls Research Articles

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

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

Suppression of cell wall stiffening along coleoptiles of wheat (Triticum aestivum L.) seedlings grown under osmotic stress conditions

Effects of polyethylene glycol (PEG)-induced osmotic stress on the mechanical properties of cell walls and the levels of their components were investigated along intact wheat (Triticum aestivum L.) coleoptiles. Stress-relaxation analysis showed that the cell walls of stressed coleoptiles were loosened as compared with those of unstressed ones not only in the apical but in the basal regions. The amounts of wall-bound ferulic acid (FA) and diferulic acid (DFA) of stressed coleoptiles were substantially lower than those of unstressed ones in all regions. The cellulose and hemicellulose contents increased toward the coleoptile base. Osmotic stress reduced the cellulose content in the basal region but it slightly affected the hemicellulose content. The molecular weight of hemicellulose in the apical region of stressed coleoptiles was higher than that of unstressed ones, while that in the basal region was almost the same in both coleoptiles. FA, DFA and cellulose contents correlated with the cell wall mechanical property. The amount and molecular weight of hemicellulose, however, did not correlate. These results suggest that the reduced levels of FA and DFA in all regions and cellulose in the basal region of wheat coleoptiles are involved in maintaining the cell wall extensibility under osmotic stress.

Gravistimulated asymmetries in the outer epidermal cell walls of graviresponding coleoptiles.

Gravitropic plant growth is due to gravistimulated asymmetric extension rates of the affected flanks of the graviresponding organ. Differential growth of the upper and lower flanks (UFs, LFs) of graviresponding plant organs may in principle be achieved by various biochemical and/or biophysical asymmetries. The gravistimulated mechanism(s) by which the different growth rates are determined is still unresolved, as is the mechanism of IAA-regulated growth. The purpose of this brief review is to summarize and critically evaluate data concerning gravistimulated asymmetries, especially with respect to the interface of the plasma membranes and of the extension-restricting, load-bearing epidermal cell walls (Masuda and Yamamoto 1972 Physiol Plant 27:109-115). In addition, recent results obtained by the author in experiments with rye coleoptiles will be presented and discussed in the context of a tentative model of gravistimulated wall asymmetries temporarily causing differential growth.

Osmotic Stress Suppresses Cell Wall Stiffening and the Increase in Cell Wall-Bound Ferulic and Diferulic Acids in Wheat Coleoptiles.

The relationship between the mechanical properties of cell walls and the levels of wall-bound ferulic (FA) and diferulic (DFA) acids was investigated in wheat (Triticum aestivum L.) coleoptiles grown under osmotic stress (60 mM polyethylene glycol [PEG] 4000) conditions. The cell walls of stressed coleoptiles remained extensible compared with those of the unstressed ones. The contents of wall-bound FA and DFA increased under unstressed conditions, but the increase was substantially reduced by osmotic stress. In response to PEG removal, these contents increased and reached almost the same levels as those of the unstressed coleoptiles. A close correlation was observed between the contents of FA and DFA and the mechanical properties of cell walls. The activities of phenylalanine ammonia-lyase and tyrosine ammonia-lyase increased rapidly under unstressed conditions. Osmotic stress substantially reduced the increases in enzyme activities. When PEG was removed, however, the enzyme activities increased rapidly. There was a close correlation between the FA levels and enzyme activities. These results suggest that in osmotically stressed wheat coleoptiles, reduced rates of increase in phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities suppress phenylpropanoid biosynthesis, resulting in the reduced level of wall-bound FA that, in turn, probably causes the reduced level of DFA and thereby maintains cell wall extensibility.

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.

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.

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.

Osmotic Stress-Induced Growth Suppression of Dark-Grown Wheat (Triticum aestivum L.) Coleoptiles

Application of 60 mM polyethylene glycol (PEG) to dark-grown wheat (Triticum aestivum L.) roots substantially reduced growth of coleoptiles. However, when PEG was removed, the growth rate of these coleoptiles greatly increased. Cell walls of stressed coleoptiles remained loosened as compared with those of unstressed ones. The osmotic potential of the stressed coleoptiles decreased to that of the 60 mM PEG solution. On the other hand, the extent of decrease in the osmotic potential of stressed roots was smaller than that of stressed coleoptiles. The osmotic potential difference (An) between the cell sap and the incubation medium of stressed roots was substantially higher than that of unstressed ones. The amount of ink moved from roots, where it was applied, to the apical region of coleoptiles was significantly reduced under osmotic stress conditions. When water was exogenously applied to abraded coleoptiles, the growth of these stressed coleoptiles was greatly promoted. These results suggest that inhibition of coleoptile growth under osmotic stress conditions is not directly related to a decrease in cell wall extensibility or to loss of the capacity to maintain osmotic potential gradients, but is caused by the reduction of the water supply from the roots to the coleoptiles.

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

Acid phosphatase isoforms in dry seeds and during seedling development in barley (Hordeum vulgare)

The acid phosphatase activity in the soluble, membrane, and cell wall fractions from Hordeum vulgare in dry seeds and during seedling development was investigated. The acid phosphatase activities were also assayed in barley roots and coleoptiles at different developmental stages. Electrophoretic patterns of multiple acid phosphatases in seeds, endosperms and embryos, and growing roots and coleoptiles are shown. The enzyme activity shows a rapid decrease in both roots and coleoptiles during growth. Using nondenaturing polyacrylamide gel electrophoresis, multiple acid phosphatase forms were found in all the organs examined. However, no qualitative differences in the location of bands were observed between root and coleoptile extract at various stages of development. The coleoptile cell wall fraction showed an acid phosphatase form characterized by a very low electrophoretic mobility that was not found in the soluble fraction. Keywords: barley, Hordeum vulgare L., acid phosphatase, isoforms, seedlings growth.

Characterization of the isozymes of glucuronoxylan xylanohydrolase in the presence of a native cell wall substrate.

A simple but accurate method for measuring glucuronoxylan xylanohydrolase activity was developed using coleoptile cell wall particles prepared from maize (Zea mays L.). Two isozymes of glucuronoxylan xylanohydrolases designated as GX1 and GX2 (EC 3.2.1.136) were purified from a commercially available amylase preparation to electrophoretic homogeneity by three cation exchange chromatography steps. Upon characterization no significant differences between the two enzymes were detected: the molecular mass measured by MALDITOF mass spectrometry was 44,360 +/- 100 for GX1 and 44,370 +/- 50 for GX2 suggesting no difference in the total number of amino acid residues. Furthermore the N-terminal amino acid sequence for each of the isozymes was identical through the 37th amino acid residue. The values of pI were determined to be 9.0 for GX1 and 9.1 for GX2. The sensitivity to temperature, pH and to ionic strength was similar for both isozymes as were kinetic parameters including Km and Vmax. No differences could be detected in substrate specificity.

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.

Characterization of Growth-related Osmiophilic Particles in Corn Coleoptiles and Deepwater Rice Internodes

Osmiophilic particles are secreted from the epidermal cells into the outer epidermal cell wall of deepwater rice internodes and corn coleoptiles that have been induced to elongate rapidly by treatment with gibberellin or auxin, respectively. The diameter of the osmiophilic particles is 80 to 100 nm in deepwater rice and up to 300 nm in corn coleoptiles. Because these particles are secreted to the outer epidermal wall only, they may contain either cell wall components that are specific for the outer epidermal wall or components of the cuticle that coats the outer epidermal wall. Monensin inhibited the appearance of these particles, indicating that they are derived from the Golgi apparatus. An attempt was made to identify the contents of the osmiophilic particles by enzyme-gold or antibody-gold labelling. Cutinase-gold accumulated mostly in the region of the cell wall just below the cuticle. The osmiophilic particles became densely labelled after incubation with proteinase K-gold, indicating that they are, at least in part, proteinaceous. In excised tissue and ultrathin sections treated with proteinase K, the number of osmiophilic particles was reduced, while glucanase and cutinase treatments were without effect. Antibodies against lipid transfer protein, an extensin-like protein, an arabinogalactan protein, a peroxidase, and expansin bound to the cell wall or plasma membrane but not to the osmiophilic particles.

Light‐induced increase in the contents of ferulic and diferulic acids in cell walls of Avena coleoptiles: its relationship to growth inhibition by light

White fluorescent light (5 W m−2) inhibited Avena coleoptile growth. Light caused in increase in minimum stress relaxation time and a decrease in extensibility (strain/load) of coleoptile cell walls. Light increased the contents of ferulic acid (FA) and diferulic acid (DFA) ester‐linked to the hemicellulose I in cell walls. These changes in the phenolic contents correlated with those of the mechanical properties of cell walls, suggesting that light stimulates the formation of DFA in hemicellulose I, making cell walls rigid, and thus results in growth inhibition. The ratio of DFA to FA was almost constant in the dark, but decreased in light, although it was almost constant in Oryza coleoptiles either in the dark or in light (Tan et al. 1992). From this fact, it is speculated that in the light condition, the formation of DFA in cell walls is limited in the step of the peroxidase catalyzed coupling reaction to produce DFA, while in the dark it is limited in the step of the feruloylation of hemicellulose I.

Mechanosensory microtubule reorientation in the epidermis of maize coleoptiles subjected to bending stress.

Plants respond to mechanical stress by adaptive changes in growth. Although this phenomenon is well established, the mechanism of the perception of mechanical forces by plant cells is not yet known. We provide evidence that the cortical microtubules subadjacent to the growth-controlling outer epidermal cell wall of maize coleoptiles respond to mechanical extension and compression by rapidly reorientating perpendicular to the direction of the effective force change. These findings shed new light on many seemingly unrelated observations on microtubule reorientation by growth factors such as light or phytohormones. Moreover, our results suggest that microtubules associated with the plasma membrane are causally involved in sensing vectorial forces and provide vectorial information to the cell that can be utilized in the orientation of plant organ expansion.

Ultrastructure of the Interface between Erysiphe pisi and Its Nonhost Plant, Barley.

The interface between Erysiphe pisi and its nonhost, barley, was investigated by transmission electron microscopy. Coleoptiles of barley inoculated with the fungus were fixed at predetermined time intervals: (1) before penetration; (2) 30min after initiation of coleoptile cytoplasmic aggregation; (3) 15-16hr after cytoplasmic aggregation. When appressoria of the fungus matured on the surface of coleoptiles, the appressoria cell wall was thin and flat where the appressorium was in contact with the coleoptile surface. At this stage of development, no noteworthy changes occurred in coleoptile cells below appressoria. In specimens fixed 30min after the initiation of cytoplasmic aggregation, short penetration pegs from appressoria, breached the coleoptile cell wall and were surrounded by and embedded with papillae. The papillae at this stage were mostly electron-dense with some heterogeneous, electron-lucent regions. A large number of spherosomes appeared around the margin of papillae in cytoplasmic aggregates. Some of spherosomes were attached to the margin of papillae and probably allowed their contents to be deposited into the papillae. In specimens fixed 15-16hr after the initiation of cytoplasmic aggregation, prominent papillae were always found below appressoria which apparently had attempted to penetrate the cell wall of coleoptile. These papillae consisted of multiple, alternately electron-dense and -lucent layers. This suggests that osmiophilic and non-osmiophilic materials were deposited alternately during papilla development which occurred in a period of 15-16hr after the initiation of cytoplasmic aggregation. Thus, the gradual development of papillae probably depends on such a continuous deposition of materials.

Determination of Auxin-Dependent pH Changes in Coleoptile Cell Walls by a Null-Point Method.

The present debate on the validity of the "acid-growth theory" of auxin (indole-3-acetic acid, IAA) action concentrates on the question of whether IAA-induced proton excretion into the cell wall is quantitatively sufficient to provide the shift in pH that is required to explain IAA-induced growth (see D.L. Rayle, R.E. Cleland [1992] Plant Physiol 99:1271-1274 for a recent apologetic review of the acid-growth theory). In the present paper a null-point method has been employed for determining the growth-effective cell-wall pH in the presence and absence of IAA after 60 min of treatment. Elongation of abraded maize (Zea mays L.) and oat (Avena sativa L.) coleoptile segments was measured with the high resolution of a displacement transducer. The abrasion method employed for rendering the outer epidermal cell wall permeable for buffer ions was checked with a dye-uptake method. Evidence is provided demonstrating that externally applied solutes rapidly and homogeneously penetrate into the epidermal wall, whereas penetration into the inner tissue walls is strongly retarded. "Titration" curves of IAA-induced and basal elongation were determined by measuring the promoting/inhibiting effect of medium pH under iso-osmotic conditions in the range of pH 4.5 to 6.0. In maize, the null point (no pH-dependent change in elongation rate after 5-10 min of treatment with 10 mmol L-1 citrate buffer) was pH 5.00 after 60 min of IAA-induced growth, and the null-point pH determined similarly in IAA-depleted tissue (10 times smaller elongation rate) was 5.25. Corresponding titration curves with Avena segments led to slightly lower null-point pH values both in the presence and absence of IAA-induced growth. After induction of acid-mediated extension by 1 [mu]mol L-1 fusicoccin (FC) in maize, the null-point pH shifted to 3.9. At 0.5 [mu]mol L-1, FC induced the same elongation rate as IAA but a 9-fold larger rate of proton excretion. At 0.033 [mu]mol L-1, FC induced the same rate of proton excretion as IAA but had no appreciable effect on elongation. The implications of these results against the background of recent attempts to revitalize the acid-growth theory of IAA action are discussed.

Effects of Extracellular Calmodulin on Cell Proliferation of Angelia dehurica Suspension Culture

1 Introduction There have been a lot of reports on the regulation of calmodulin in the cell proliferation of animal cell. Crocker et al. fast presented the experimental evidence which showed that calmodulin might accelerate human leukaemic lymphocytes proliferation.Biro et al. reported that in plants calmodulin was presented in the cell wall of wheat coleoptiles. Then it was confirmed by several experiments, especially the purification of

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.