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; )     

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.     

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.     

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.     

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.     

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

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

Force extension analysis of Avena coleoptile cell walls

Summary A method is described for measuring the cell wall mechanical properties of Avena coleoptiles in the absence of turgor stress or influences of a living protoplast. Forceextension curves obtained with a constant-rate-of-extension instrument and standard fiber-testing techniques demonstrate the permanence of cell wall loosening effects of prior indoleacetic acid (IAA) treatment of living tissue and provide evidence that these changes involve interactions between cell wall polymers. By this method various chemical and enzymatic modifications of cell walls can be evaluated in terms of altered mechanical properties. Thus, it was possible to remove over 97% of the cell nitrogen (including some hydroxyproline-containing protein) by hot methanol followed by enzymatic treatment and not change the extensibility properties of the tissue. In contrast, coleoptile mechanical properties were markedly influenced by chemical acetylation procedures or cellulase treatment.With 3 Figures in the Text     

Auxin-induced changes in the molecular weight of hemicellulosic polysaccharides of the Avena coleoptile cell wall

The average molecular weight of the water soluble hemicelluloses(hemicellulose B) of the Avena coleoptile cell wall was determinedby gel permeation chromatography (GPC) and viscometry. Analysisof the neutral sugar composition of henucellulose B eluted froma GPC column (Sepharose 4B) indicated that it consists of ß-glucanwith a high molecular weight and arabinoxylan with a low molecularweight. A kinetic study of the effect of auxin on the moleculardistribution of henucellulose B demonstrated that auxin decreasedthe ß-glucan content of the hemicellulose as earlyas the first hour incubation, but not the arabinoxylan content,when it stimulated the extension of the coleoptile segments.Calculation of the weight-average molecular weight from thechromatograms suggested that auxin decreased the molecular weightof hemicellulose B; this was also confirmed by viscometry. Thus,auxin may cause cell wall loosening, leading to cell extension,through its effect on ß-glucan degradation or throughthe decrease in the molecular weight of hemicellulose B. (Received July 16, 1979; )     

The determination of sensitivity parameters for auxin-induced H+-efflux from Avena coleoptile segments

Abstract A method is described for the measurement of auxin-induced H⁺-efflux from small populations of Avena coleoptile segments. The method allows the simultaneous investigation of the kinetics of rapid auxin responses over a wide concentration range. IAA promoted linear rates of H⁺-efflux and the change in amplitude of response occurred mainly over a low, narrow concentration range (10–50 μmol m^-3). The sigmoidal curve of best-fit to each set of dose-response data was determined using non-linear regression techniques, allowing the objective determination of characteristic tissue sensitivity parameters (R_MIN, R_MAX, K_D and p). The sensitivity parameters for the auxin-type herbicide fluroxypyr are also presented as well as IAA parameters determined in the presence of abscisic acid and the ‘antiauxin’ PCIB. The interpretation of the parameter values and the potential use of sensitivity parameter analysis for the evaluation of theories concerning plant hormone action and interaction is discussed.     

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.     

The effect of ethylene on auxin-induced growth of excised rice coleoptile segments

Elongation growth induced by exogenous auxin of apical coleoptilesegments of etiolated rice seedlings was promoted by ethylene.In the absence of exogenous auxin, growth promotion was notobserved. The highest promotion by ethylene was obtained at10^–6 M of indole-3-acetic acid, a suboptimal concentrationfor auxin-induced elongation. Level of ethylene which achievedthe effect was less than 1 µl per liter of an incubationatmosphere. ¹Present address: The Ocean Research Institute, University ofTokyo, Nakano, Tokyo, Japan (Received May 27, 1970; )     

Activation of Avena coleoptile cell wall glycosidases by hydrogen ions and auxin

Several cell wall-bound glycosidases present in Avena sativa coleoptiles were assayed by following the hydrolysis of p-nitrophenyl-glycosides. Particular emphasis was placed on characterizing some parameters affecting the activity of β-galactosidase. The pH optimum of this enzyme is 4.5 to 5.5; it is sensitive to copper ions and p-chloromercuribenzoate treatment and apparently has an exceptionally low turnover rate. Indoleacetic acid treatment enhanced in vivo β-galactosidase activity of coleoptile segments by 36% over control after 60 minutes. This enhancement was prevented by abscisic acid and cycloheximide. High buffer strengths and low pH reduced the indoleacetic acid-enhanced increase in enzyme activity. These data lend support to the following proposed model of indoleacetic acid action. Indoleacetic acid enhances the release of hydrogen ions into the cell wall which promote the activities of cell wall glycosidases, some of which may participate in the cell extension process.     

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.     

Protein synthesis and wall extensibility in the Avena coleoptile

Summary The inhibitors cycloheximide and puromycin have been used to examine the relationship between protein synthesis and wall extensibility, as measured with an Instron, in Avena coleoptile segments. Cycloheximide at 4 g/ml almost totally inhibits both auxin-induced cell elongation and protein synthesis with only a slight lag. Wall extensibility is unaffected by the inhibitor if auxin is absent. If added prior to auxin, cycloheximide prevents auxin-induced wall loosening while if added after auxin it causes a substantial decline in the wall extensibility. With puromycin there is a 2–4 hr lag before growth and wall loosening are inhibited. These results support the conclusions that the proteins needed for wall loosening are unstable, and that continued protein synthesis is necessary to maintain the wall loosening process.     

Absence of auxin-induced stored growth in Avena coleoptiles and its implication concerning the mechanism of wall extension

Summary We have reinvestigated the ability of Avena coleoptiles to undergo auxin-induced stored growth (stored growth is defined as the ability of a cell to store up a potential for extension during periods of reduced turgor which can be converted into extra extension upon restoration of normal turgor). We could detect little or no stored growth, with either moderate (1–2 bar) or more severe (3–5 bar) reductions in turgor, and with varying periods (10–100 min) of reduced turgor. Earlier reports of a stored growth potential (e.g., Cleland and Bonner, 1956) are shown to be in error, in that the apparent growth potential is probably an artifact of the use of argon or nitrogen as an inhibitor of auxin action. The absence of stored growth reported here is not due to a direct inhibitory effect of the osmoticum itself on auxin action, since coleoptiles can extend in response to auxin even in the presence of mannitol if an external force is applied to the section to replace the normal turgor. These results show that the two components of cell-wall extension, wall loosening and wall extension, usually are inseparable. Two possible explanations are considered; the walls may be extending by the process of chemical creep, or the wall loosening may only occur when the load-bearing bonds are under tension.     

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; )     

UDP-Glucose level as a limiting factor for IAA-induced cell elongation in Avena coleoptile segments

The effects of galactose on IAA-induced elongation and endogenous level of UDP-glucose (UDPG) in oat ( Avena sativa L. cv. Victory) coleoptile segments were examined under various growth conditions to see if there was a correlation between the level of UDPG and auxin-induced growth. The following results were obtained:

• (1)

  Galactose (10 m M ) inhibited the auxin-induced cell elongation of oat coleoptile segments after a lag of ca 2 h. Determinations of cell wall polysaccharides and UDP-sugars indicated that galactose, when inhibiting the cell wall polysaccharide synthesis, decreased the level of UDPG but caused an increase in the levels of Gal-1-P and UDP-Gal.
• (2)

  When coleoptile segments treated with IAA and galactose were transferred to galactose-free IAA-solution, the segment elongation was restored and the amounts of cell wall polysaccharides increased. During this period, the amount of UDPG increased and the levels of Gal-1-P and UDP-Gal slightly decreased or leveled off. The UDP-pentoses changed similarly as UDPG did.
• (3)

  Addition of sucrose (30 m M ) enhanced IAA-induced cell elongation and removed growth inhibition by 1 m M galactose. Sucrose increased the amounts of the cell wall polysaccharides and the level of UDPG in the presence or absence of IAA and also counteracted the decrease in UDPG caused by galactose.

These results indicate that the level of UDPG is an important limiting factor for cell wall biosynthesis and, thus, for auxin-induced elongation.     

Effect of indole-3-acetic acid on cell wall loosening: Changes in mechanical properties and noncellulosic glucose content of Avena coleoptile cell wall

The effect of indole-3-acetic acid on cell wall loosening andchemical modifications of noncellulosic components of the cellwall in Avena coleoptile segments was studied and the followingresults were obtained. (1) Auxin decreased both the minimum stress-relaxation time(T_o) and the noncellulosic glucose content of the cell wall. (2) Decreases were observed in the absence or presence of mannitolsolution at concentrations lower than 0.20 M which osmoticallysuppressed auxin-induced extension, while at concentrationshigher than 0.25 M, there was little auxin effect, indicatingthat it is turgor-dependent. (3) The decrease in T_o of the cell wall and that in the noncellulosicglucose content caused by auxin in the presence of mannitolsolutions of various concentrations paralleled each other (thecorrelation coefficient was 0.897). (4) Both decreases in T_o and glucose content caused by auxinwere inhibited by nojirimycin (5-amino-5-deoxy-D-glucopyranose)in the presence of mannitol. The results suggest that auxin-induced cell wall loosening iscaused by the degradation of noncellulosic rß-glucanin the cell wall. (Received December 24, 1976; )