Wounding-induced cell wall pH shifts in coleoptile segments of various Poaceae

Wounding-induced extracellular pH shifts were characterized previously in excised segments of maize (Zea mays L.) coleoptiles. In the present study it is demonstrated that similar pH shifts also occur in Triticum aestivum L., Secale cereale L., Hordeum vulgare L., Avena sativa L., Sorghum durra (Forsk.) Stapf, and Setaria italica (L.) Beauv., with characteristic quantitative differences between the species. Indole-acetic acid induces pronounced drops of the medium pH in all species except Setaria italica. This revised version was published online in July 2006 with corrections to the Cover Date.     

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; )     

Auxin-induced extension, cell wall loosening and changes in the wall polysaccharide content of barley coleoptile segments

Contents of the cell wall and sugar pool and the response toexogenously applied auxin (cell extension and cell wall loosening)were investigated with barley coleoptile segments excised from4-, 5- and 6-day-old seedlings. The first two groups exhibiteda high capacity to grow in terms of the intact growth rate andwere responsive to auxin, while those excised from 6-day-oldseedlings had a low growth capacity. The cell wall of 4- and5-day-old coleoptile segments contained almost the same amountof noncellulosic wall components per unit length while the 6-day-oldones had a lesser amount. The sugar pool and -cellulose contentper unit length decreased as the coleoptile aged. Auxin-stimulatedextension was most marked in the 4-day-old coleoptile segments.Auxin caused quantitative changes in the cell wall componentsof 4-day-old coleoptiles and, to a lesser extent, of 5-day-oldcoleoptiles, i.e., an increase in the contents of xylose andarabinose, both of which are constituents of noncellulosic polysaccharidesof the cell wall, and of -cellulose and a decrease in the noncellulosicglucose content. Auxin caused very little change in the noncellulosicsugar content and -cellulose content of the cell wall from 6-day-oldcoleoptile segments. The auxin-induced change in mechanicalproperties of the cell wall was significant in 4- and 5-day-oldcoleoptiles but very small in 6-day-old ones. The results suggestedthat the content of noncellulosic wall components is closelyrelated to the intact growth and auxin responsiveness of barleycoleoptiles. (Received April 20, 1978; )     

Brefeldin A: a specific inhibitor of cell wall polysaccharide biosynthesis in oat coleoptile segments

The effect of brefeldin A (BFA) on the synthesis and incorporation of polysaccharides, proteins and glycoproteins into the cell wall of subapical coleoptile segments isolated from etiolated oat seedlings (Avena sativa L. cv. Angelica) has been investigated. In the presence of D-[U-¹⁴C]-glucose, the incorporation of radioactive glycosyl residues into buffer-soluble, membrane (matrix polysaccharides) and cell wall polysaccharides was drastically inhibited by increasing concentrations of BFA up to 10 μ·mL⁻¹. BFA also altered the pattern of these polysaccharides suggesting a different sensitivity of glycosyltransferases toward the action of the drug. The incorporation of [U-¹⁴C]-glycine or L-[U-¹⁴C]-leucine into non-covalently- and covalently-bound cell wall proteins as well as the incorporation of radioactive N-acetylglucosamine residues into the newly synthesised oligosaccharidic chains of cytosolic, membrane and cell wall glycoproteins remained unchanged in the presence of 10 μg·mL⁻¹ BFA. The data demonstrate that, in oat coleoptile segments, BFA specifically inhibits the synthesis of cellulose and matrix polysaccharides without altering the synthesis and incorporation of proteins and glycoproteins into the cell wall. In addition, it is demonstrated that BFA does not affect the in vivo activity of glycosyltransferases involved in the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the oligosaccharidic chains of glycoproteins.     

Effect of osmotic shock on auxin-induced cell extension, cell wall changes and acidification in Avena coleoptile segments

The effect of plasma membrane alteration caused by osmotic shockof different strengths on the auxin-induced responses of Avenacoleoptile cells was observed. Osmotic shock brought about by0.5–0.7 M mannitol solution for 10 or 30 min, followedby phosphate-buffer (1 mM, pH 6.0) treatment for 10 min at 4?Ccaused no significant inhibition of auxin-induced cell extension.The osmotic shock did not affect auxin-induced cell wall looseningrepresented by stress-relaxation time and a decrease in thenoncellulosic glucose level of the cell wall. The shock causedonly a temporary inhibition of transmembrane potential and noinhibition of oxygen consumption. However, it inhibited auxin-stimulatedH⁺ secretion which was reversed by 0.1 mM CaCl₂. We concludedthat the Osmotic shock may partly modify the plasma membranerelated to the hydrogen ion pump which interacts with auxin,but this modification which is reflected little by the transmembranepotential and cellular metabolism, is not closely related toauxin-induced cell wall loosening and thus cell extension inAvena coleoptiles. ³ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan (Received February 17, 1978; )     

Phenolic acids in wheat coleoptile cell walls

The phenolic constituent of nonvascular cell walls of wheat (Triticum aestivum L.) coleoptiles, which yields vanillin upon nitrobenzene oxidation, is not lignin as I previously claimed. It seems to be mainly ferulic acid bonded to carbohydrate, probably by an ester linkage. The acid is associated with a fraction of the wall rich in arabinose and xylose, although it is not known whether it is esterified directly with these pentose residues. The phenolic-carbohydrate complex is released by cellulase, but not by pronase or a mixture of hemicellulases.     

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.     

Auxin-indnced changes in barley coleoptile cell wall composition

Auxin induces extension growth of barley coleoptile segments,causing cell extension and cell wall loosening represented bya change in mechanical properties of the cell wall. This responsedecreased after the segments were starved for more than 12 hrin buffer solution. Auxin decreased the noncellulosic glucosecontent of the cell wall of the segments starved for 0 and 6hr, but very little that of segments starved for 12 and 18 hr.The contents of arabinose, xylose and galactose, among noncellulosicpolysaccharides, and -cellulose of the cell wall increased duringthe starvation, but auxin did not affect them. The auxin-induceddecrease in glucose content was inhibited by nojirimycin, apotent inhibitor of ß-glucanase, which inhibited auxin-inducedextension and changes in mechanical properties of the cell wall,suggesting that cell wall loosening, and thus cell extension,resulted from partial degradation of ß-glucan of thecell wall. (Received April 20, 1978; )     

Inhibition of the synthesis of cell wall polysaccharides in oat coleoptile segments by jasmonic acid: Relevance to its growth inhibition

The inhibitory mode of action of jasmonic acid (JA) on the growth of etiolated oat (Avena sativa L. cv. Victory) coleoptile segments was studied in relation to the synthesis of cell wall polysaccharides using [¹⁴C]glucose. Exogenously applied JA significantly inhibited indoleacetic acid (IAA)-induced elongation of oat coleoptile segments and prevented the increase of the total amounts of cell wall polysaccharides in both the noncellulosic and cellulosic fractions during coleoptile growth. JA had no effect on neutral sugar compositions of hemicellulosic polysaccharides but substantially inhibited the IAA-stimulated incorporation of [¹⁴C]glucose into noncellulosic and cellulosic polysaccharides. JA-induced inhibition of growth was completely prevented by pretreating segments with 30 mm sucrose for 4 h before the addition of IAA. The endogenous levels of UDP-sugars, which are key intermediates for the synthesis of cell wall polysaccharides, were not reduced significantly by JA. Although these observations suggest that the inhibitory mode of action of JA associated with the growth of oat coleoptile segments is relevant to sugar metabolism during cell wall polysaccharide synthesis, the precise site of inhibition remains to be investigated.Abbreviations JA jasmonic acid - ABA abscisic acid - IAA indoleacetic acid - T ₀ minimum stress relaxation time - TFA trifluoroacetic acid - TCA trichloroacetic acid - HPLC high-performance liquid chromatography - EtOAc ethyl acetate - TLC thin-layer chromatography - JA-Me methyl jasmonate - GLC-SIM gas-liquid chromatography-selected ion monitoring     

Growth and cell wall changes in rice coleoptiles growing under different conditions II. Auxin-induced growth in coleoptile segments

The effect of auxin on growth, mechanical properties of thecell wall and cell wall sugar composition in rice coleoptilesegments excised at different ages from seedlings growing underdifferent conditions were investigated. Auxin markedly inducedgrowth only in segments excised from coleoptiles in the fastgrowth phase with a high content of non cellulosic glucose intheir cell walls. The response to auxin decreased with coleoptileage, accompanying a decrease in the amount of the noncellulosicglucose in the cell wall, suggesting a correlation between noncellulosicglucose content and growth capacity in response to auxin. Goodcorrelation among auxin-induced growth, auxin-induced decreasein the T_o value and auxin-induced decrease in the noncellulosicglucose content of the cell wall also was found. ¹ Present address: Departamento Fisiologia Vegetal, Facultadde Ciencias, Universidad de Salamanca, Salamanca, Spain. (Received May 21, 1979; )     

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.     

Transient changes in length and growth of wheat coleoptile segments following treatments with osmotica and auxin

The dependence of elongation on the osmotic potential of the medium was investigated, using coleoptile segments (CS) of Triticim aestivum L. (cv. Hartri) and an optoelectronic device. The study aimed at separating the osmoelastic response from the irreversible growth response when an osmoticum (mannitol) was added, and to compare both processes in order to consider the possibility of growth-induced reduction in turgor pressure. The prompt inhibition of elongation registered just after addition of 50 mM mannitol as well as the subsequent resumption of the original elongation rate could be quantitatively explained by the extent and the kinetics of the osmoelastic relaxation. An initial reduction in the irreversible elongation component by mild osmotic stress could not be demonstrated. Above a critical value, the irrevesible growth was insensitive to a further increase in water potential. The minimum turgor pressure required to drive steady growth was not far from zero in both the presence and absence of auxin. The rate (r) of osmotically caused shortening per unit change of water potential was determined from the kinetics of CS shortening induced by addition of mannitol at nearly isotonic concentration (300 mM). This parameter relates a fractional change in length to the difference in water potential between inside and outside, and was assumed to depend largely on the hydraulic resistance of the tissue and cuticle. It was found to be independent of IAA. The relatively low value of Γ suggests significant reduction of turgor at high growth rates. In accordance with this conclusion, the extent of osmoelastic shortening after a transfer to 300 mM mannitol (dependent on wall strain) was significantly decreased in the presence of IAA. Addition of 100 μM IAA to CS growing at a constant rate induced pronounced oscillations in the rate of elongation, which may be connected with the change in elastic cell wall strain. Whereas the steady state growth rate before the addition of IAA was the same in the presence and in the absence of 50 mM mannitol, the maximum growth rate found after addition of IAA was substantially reduced in the mannitol variant.     

Effect of galactose on auxin-induced cell elongation in oat coleoptile segments in mannitol solutions

Oat coleoptile segments were treated with or without 10 mM galactose in the presence or absence of 10 μM IAA and various concentrations of mannitol (pre-incubation). Auxin-induced growth was inhibited by galactose. Segments were then transferred to buffer solutions containing or not containing 10 mM galactose (post-incubation). Expansion growth due to rapid water absorption was observed. The expansion growth during the post-incubation was inhibited by galactose when galactose was applied during the post-incubation period or all through the pre- and post-incubation but was not affected by galactose when it was applied only during the pre-incubation. This result indicates that the galactose effect on the expansion growth is due to its inhibitory action during the post-incubation period. Galactose has been reported to be a specific inhibitor for cell wall synthesis. Thus, it is suggested that the expansion growth during post-incubation requires cell wall synthesis and is not just the process of passive water absorption. The primary action of auxin does not seem to require new synthesis of polysaccharides.     

The mechanical behavior of isolated Avena coleoptile walls subjected to constant stress: properties and relation to cell elongation

In order to assess the role of the mechanical properties of the wall in auxin-induced cell elongation, a study has been made of the ability of isolated Avena coleoptile walls to extend (creep) when subjected to a constant applied stress. Creep occurs as a viscoelastic extension which has the following characteristics: the extension is proportional to log time and is partly reversible, and the extension rate has a Q₁₀ of about 1.05 and is markedly greater in auxin-pretreated walls. In nonconditioned walls the extension rate is proportional to applied stress, but pre-extension causes the appearance of an apparent yield strain. The similarity of creep and instantaneous plastic deformation in response to temperature or to pretreatment with auxin or KCN suggests that the instantaneous deformation is simply the viscoelastic extension which occurs at very short times. A comparison of these viscoelastic properties with the properties of auxin-induced cell elongation indicates that cell elongation requires more than just a physical extension of the wall. It is suggested that elongation occurs as a series of extension steps, each of which involves a viscoelastic extension preceded or accompanied by an auxin-dependent biochemical change in the wall properties.     

The coleoptile sheath of wheat impedes oxygen transport to seminal roots

Abstract Wheat, Triticum aestivum cv. Gamenya, seedlings were grown for 4d in acrated 0.5 mol m^?3 CaSO₄ solution. A cylindrical platinum-electrode and vernier microscope were used to examine the effects of the colcoptile sheath on O₂ transport from shoots to seminal roots via the internal gas-space system, and on the clongation of the root tip. By removing the colcoptile sheath, O₂ concentrations at the root tip in an O₂-free medium at 5°C increased from 0.017 to 0.17 mol m^?3 O₂ when 100 kPa O₂ was around the shoots. When the shoots were in air, the separation of the colcoptile sheath from the primary leaf caused the rate of root clongation in the anoxic medium to increase by two fold.     

The nature of spontaneous changes in growth rate in isolated coleoptile segments

About 4 hours after they are cut from the seedling, corn (Zea mays L.) coleoptile segments mounted vertically show a strong increase in growth rate. This increase occurs in water or various buffers near pH 7 and is not accompanied by the accumulation of a growth promoter in the medium. The increase in growth rate is prevented by 1 mmp-fluorophenylalanine and is strongly inhibited by 0.1 mmp-chlorophenoxyisobutyric acid.The increased growth rate is accompanied by a 95% increase in the ability of tissue extracts to catalyze the conversion of (14)C-tryptophan to (14)C-indole-3-acetic acid and by a nearly 3-fold increase in indole-3-acetic acid oxidase activity. The increase in growth rate is also observed in segments from coleoptiles grown aseptically.The spontaneous increase in growth rate is completely but reversibly inhibited by 1 mum indole-3-acetic acid. Cytokinins have little effect on the spontaneous growth response, whereas gibberellic acid is observed to extend the latent period and reduce the magnitude of the response. It is tentatively concluded that the increase in endogenous growth rate may result from increased auxin production upon derepression of the auxin biosynthesis pathway after isolating the tissue from the normal supply of auxin from the tip.     

Effect of lectins on auxin-induced elongation and wall loosening in oat coleoptile and azuki bean epicotyl segments

A marine unicellular aerobic nitrogen-fixing cyanobacterium Synechococcus sp. strain Miarni BG 043511 was pretreated with different light and dark regimes in order to induce higher growth synchrony. A pretreatment of two dark and light cycles of 16 h each yielded good synchrony for 3 cell division cycles. Longer dark treatments decreased the degree of synchrony and shorter dark treatments caused irregular cell division. Once synchronous culture was established, distinct phases of cellular carbohydrate accumulation and cellular carbohydrate degradation were observed even under continuous illumination. Changes in carbohydrate content were repeated in a cyclic manner with approximately 20 h intervals, the same as the cell division cycle. This change in carbohydrate metabolism provided a good index of growth synchrony under nitrogen-fixing conditions.
Photosynthetic oxygen evolution and nitrogen fixation capabilities and their activities in near, in situ, culture conditions were measured in well synchronized cultures of this strain under continuous illumination. Distinct oscillations of both photosynthetic oxygen evolution and nitrogen fixation capabilities with ca 20-h intervals, similar to the interval of the cell division cycle, were observed for three cycles. However, the activities of photosynthetic oxygen evolution were inversely correlated with those of nitrogen fixation. During the nitrogen fixation period, net oxygen consumption was observed even in the light under conditions approximating in situ culture conditions. The phase of temporal appearance of nitrogenase activity during the cell division cycle coincided with the phase of carbohydrate net degradation. These data indicate that this unicellular cyanobacterium can grow diazotrophically under conditions of continuous illumination by the segregation of photosynthesis and nitrogen fixation within a cell division cycle.     

Lignin formation in wheat coleoptile cell walls: a possible limitation of cell growth

Four growth-influencing compounds—hydroxyproline, 2,2′-dipyridyl, 2-chloroethylphosphonic acid, and indoleacetic acid—were used to examine the relationship between lignin formation and growth of wheat coleoptile sections. Hydroxyproline and 2-chloroethylphosphonic acid, at low concentrations, inhibited growth and increased lignin content. Dipyridyl, which promoted coleoptile elongation, decreased lignin content. Indoleacetic acid caused a 300% increase in growth at 0.1 mm but resulted in lignin content no different from controls with no auxin. Chemical and anatomical evidence is given which indicates that lignin is present in the epidermal cell walls of the wheat coleoptile. It is thus possible that bonding between lignin and hemicellulose may have some influence on coleoptile growth.     

Membrane-bound glucosamine acetyltransferase in coleoptile segments of Avena sativa

The in vivo metabolism of D-[U-¹⁴C]glucosamine and the in vitro properties of glucosamine acetyltransferase (EC 2.3.1.3), the first committed enzyme in the metabolism of exogenously supplied D-glucosamine, were studied in coleoptile segments of Avena sativa L. cv. Sole II. D-[U-¹⁴C]glucosamine was taken up by oat coleoptile segments and sequentially metabolised to radioactive N-acetylglucosamine, N-acetylglucosamine 6-P, N-acetylglucosamine 1-P, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine. In addition, N-acetylglucosamine residues were incorporated into glycoproteins and glycolipids of the cells. All glucosamine acetyltransferase activity was found to be membrane-bound. The enzyme was solubilized by either digitonin or CHAPS. The specificities and the kinetics of the membrane-bound and soluble glucosamine acetyltransferase were determined. The effects of ions, nucleotides, nucleoside diphosphate amino sugars, coenzymes and group-specific chemical probes on the rate of membrane-bound and CHAPS-solubilized enzyme were investigated. Our data indicate that UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine do not exert a feed-back control on the glucosamine acetyltransferase either in vivo or in vitro. Further, some nucleotides and the metal ions Cu²⁺, Zn²⁺, Fe²⁺, Fe³⁺ and Co²⁺ affect the activity of the enzyme in vitro.     

Response of excised Avena coleoptile segments to red and far-red light

Summary In seeking a simple, red light-promoted straight growth test in which phytochrome assays may be conducted without interference by protochlorophyll, the response of excised Avena coleoptile segments to red and far-red light was re-examined. The elongation of apical (non-decapitated) segments is promoted by a brief exposure to red light, and this effect is almost completely nullified by an immediately subsequent exposure to far-red light. Although growth promotion by red light occurs in distilled water alone, the effect is greater on a medium consisting of 0.02 M phosphate buffer, pH 6.2 to 6.4, with 1 to 2% sucrose. Over the pH range 4.5 to 7.4, dark-growth decreases with increasing pH, but the absolute increment brought about by red light is nearly constant. Elongation appears to be entirely the result of increased cell size.Contrary to previous reports, similar results can be obtained with subapical (decapitated) coleoptile segments, although the absolute magnitude of the response is reduced.Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commision.