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; )     

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 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; )     

Growth capacity in response to auxin of coleoptile segments of normal and dwarf rice strains

Auxin-induced growth, epidermal cell length, cellular osmotic potential, and cell wall composition of coleoptile segments excised from one normal and two dwarf rice strains were studied 2, 3, 4, and 5 days after soaking. The auxin-induced growth was higher at the early stages of coleoptile growth and decreased with age, being always higher in normal than in the two dwarf strains. A good correlation between auxin-induced growth and auxin-induced decrease in the minimum stress-relaxation time has been found, suggesting that the different growth capacity in response to auxin among the three different strains is due to differences in the structure of their cell walls. In fact, cell wall analysis revealed that (1) the relative -cellulose content of the cell walls was higher in the two dwarf strains than in the normal one, and (2) the auxin-induced decrease in noncellulosic glucose was high, compared with dwarf strains, in the normal strain, which showed the higher auxin-induced growth, showing a highly significant correlation between the decrease in noncellulosic glucose and the growth in response to auxin. Thus, the different growth between normal and dwarf strains might be attributed to their different capacity to degrade -glucan of their cell walls.     

Growth capacity in response to auxin of coleoptile segments of normal and dwarf rice strains

Auxin-induced growth, epidermal cell length, cellular osmotic potential, and cell wall composition of coleoptile segments excised from one normal and two dwarf rice strains were studied 2, 3, 4, and 5 days after soaking. The auxin-induced growth was higher at the early stages of coleoptile growth and decreased with age, being always higher in normal than in the two dwarf strains. A good correlation between auxin-induced growth and auxin-induced decrease in the minimum stress-relaxation time has been found, suggesting that the different growth capacity in response to auxin among the three different strains is due to differences in the structure of their cell walls. In fact, cell wall analysis revealed that (1) the relative α-cellulose content of the cell walls was higher in the two dwarf strains than in the normal one, and (2) the auxin-induced decrease in noncellulosic glucose was high, compared with dwarf strains, in the normal strain, which showed the higher auxin-induced growth, showing a highly significant correlation between the decrease in noncellulosic glucose and the growth in response to auxin. Thus, the different growth between normal and dwarf strains might be attributed to their different capacity to degrade β-glucan of their cell walls.     

The Effects of Auxin on the Mechanical Properties in Vivo of Cell Wall in Hypocotyl Segments from Gibberellin-Deficient Cowpea Seedlings

Auxin-induced changes in the mechanical properties of cell wallwere examined by both positive and negative pressure jump methodsusing hypocotyl segments excised from the 3-day-old seedlingsof cowpea that has been treated with uniconazole, a potent inhibitorof the biosynthesis of gibberellins. In such segments (U-segments)that were deficient in endogenous gibberellin, auxin increasedonly the effective turgor (Pⁱ–Y) and did not change theextensibility () of cell wall. As a result, the extent of theauxin-induced promotion of growth was halved. However, auxinwas able to increase of U-segments that has been pretreatedfor two hours with GA₃ prior to the application of IAA. Measurementof intracellular pressure (P_i) with a pressure probe revealedthat auxin did not change Pⁱ in either U-segments or GA₃-pretreatedsegments. The results suggest that auxin can decrease the yieldthreshold of the cell wall (Y) independently of gibberellinbut can increase only in the presence of gibberellin. The differencebetween and Y in terms of their requirement for gibberellinto respond to auxin suggests that they are mutually separablemechanical properties that originate from different molecularprocesses that occur in the architecture of yielding cell walls. ³Present address: Ohishi, Enden, Mori-machi, Shuchi-gun, Shizuoka,437-02 Japan     

Effects of Gibberellin on Auxin-Induced Hyperpolarization of Cell Membranes in Hypocotyls of Vigna unguiculata

Auxin activates pumping of protons from the symplast to theapoplast and causes hyperpolarization of the symplast membranein the elongation zone of Vigna stems prior to the accelerationof growth. This auxin-induced hyperpolarization has been studiedin most cases in hypocotyl segments excised from the elongationzone. In the present study, mature-zone segments were perfusedwith IAA by the xylem perfusion technique in an effort to determinewhether or not IAA has any effects in the mature zone. Althoughno hyperpolarization of the symplast membrane was observed uponthe perfusion with auxin alone, auxin-induced hyperpolarizationwas observed when mature-zone segments had been pretreated withGA₃, in the absence of an increase in the growth rate. Theseresults suggest that cells in the mature zone have lost theability to activate the proton-pumping machinery in responseto auxin but that this ability can be restored by treatmentwith GA₃. This effect of GA₃ suggests the possibility that theconcentration of gibberellin in a tissue controls one of thecell's responses to auxin, namely, activation of the protonpump. (Received January 10, 1994; Accepted June 11, 1994)     

Possible Role of Hexosamine-Containing Cell Wall Component in Growth Regulation of Rice Coleoptiles

The cell wall of rice coleoptile was found to contain severalhundred microgram hexosamine per gram dry wt with the pectic,hemicellulosic, and -cellulose fractions containing 50%, 40%,and 10%, respectively. The cell wall hexosamine content increasedseveralfold with coleoptile growth and was higher in air-typecoleoptiles (grown on the surface of water) than water-typeones (grown under water). Rice coleoptiles were cultured in glucosamine, NH₄⁺, glutamine,or asparagine solution and growth was inhibited at 10^–4M and above. Coleoptile growth capacity in glucosamine or NH₄⁺solution correlated inversely with the cell wall hexosaminecontent. Both of these solutions also inhibited elongation ofsubmerged air-type coleoptile sections. Azaserine promoted thegrowth of both intact and excised coleoptiles at 10^–6to 10^–5 M and halved the cell wall hexosamine contentof intact ones. 6-Diazo-5-oxo-L-norleucine promoted the elongationof sections. These results suggest that the hexosamine-containingcell wall component is an important growth suppression factorin rice coleoptiles. (Received April 25, 1983; Accepted August 30, 1983)     

Calcium Antagonist TMB-8 Inhibits Cell Wall Formation and Growth in Pea

The effects on auxin-stimulated growth and cell wall formationof 8-(N, N-diethylamino)-octyl-3, 4, 5-trimethoxybenzoate.HCI(TMB-8), an intracellular Ca²⁺ antagonist, were investigatedin abraded stem segments from aetiolated seedlings of Pisumsativum L. cv. Alaska. Incubation of segments at pH 6.0 with200 mmol m^–3 TMB-8 resulted in a 50% inhibition of auxin-stimulatedgrowth. Added Ca²⁺ did not restore normal auxin-stimulated growth,presumably because of its well-known stiffening effect on thecell wall. In segments incubated at a pH (7–2) which preventedelongation, auxin promoted the incorporation of [³H]glucoseinto the cell wall relative to total uptake of label. TMB-8abolished about 60% of the total incorporation of label intocell walls in the presence of auxin, but was not effective inthe absence of auxin. Exogenous CaCl₂ reversed the inhibitoryeffect of TMB-8 on relative cell wall incorporation in a parabolicmanner, with a 50% reversal at about 100 mmol m^–3 andcomplete reversal at 1.0 mol m^–3 Ca²⁺. Other ions tested(Mg²⁺, Mn²⁺, Cu²⁺, Zn²⁺) were without substantial effect atconcentrations of 0.5 mol m^–3. Both apparent uptake ofCa²⁺ and consequent reversal of TMB-8 inhibition of cell wallincorporation were blocked by the Ca²⁺ channel blockers verapamiland La³⁺. The data provide further evidence that auxin-stimulatedgrowth is dependent upon continued cell wall incorporation,and suggest that a Ca²⁺ messenger system may be involved inthe promotory actions of auxin on cell wall synthesis and long-termgrowth. Key words: Auxin, calcium, cell wall synthesis     

NaCl-and Gibberellic Acid-induced Changes in the Content of Auxin and the Activities of Cellulase and Pectin Lyase During Leaf Growth in Rice (Oryza sativa)

The interactive effect of NaCl salinity and gibberellic acidin the activities of cellulase and pectin lyase, and on thecontent of auxin and chlorophyll, has been determined duringleaf growth (fifth from base) in rice. The linear growth, chlorophyllcontent, activity of cellulase, and the auxin level of leaveswere markedly decreased when plants were exposed to salt stress(12 dS m^–1). However pectin lyase activity did not registerany significant alteration in the leaves of salt-stressed plantscompared with the control. Treatment of plants with gibberellicacid (GA₃) (10 ppm) increased the leaf growth and chlorophyllcontent with a concomitant rise in the activity of cellulaseunder stressed as well as non-stressed conditions. A markedincrease in the content of auxin was discernible in the leavesof salt-stressed plants treated with GA₃ compared with non-treatedsalinized ones. An appreciable increment in the activity ofpectin lyase in response to GA₃ administration was detectedonly in the leaves of non-stressed plants. These results indicatethat enhancement of cellulase activity and the augmentationof endogenous auxin content may be involved in the stimulationof rice leaf growth by GA₃ under saline conditions. Oryza sativa, rice, leaf growth, NaCl salinity, gibberellic acid, cellulase, pectin lyase, auxin     

The Effect of Triacontanol on Peroxidase, IAA, and Plant Growth in Pisum sativum var.'Alaska' and 'Little Marvel'

The present study investigates the effects of triacontanol (CH₃(CH₂)₂₈CH₂OH),on plant growth (root and stem), peroxidase activity (apicalmeristem tissue), and auxin destruction (apical meristem tissue)in ‘Little Marvel’ dwarf (LM) and ‘Alaska’peas (AP). Triacontanol inhibited root growth in LM comparedto untreated controls. However, root growth in AP tissue wasenhanced by 1.0 mg I^–1 triacontanol and inhibited by allother treatments, in comparison to untreated controls. Wateruptake in triacontanol-treated AP plants was greater than inuntreated controls, with the converse being the case for LM.Triacontanol treatment caused an increase in peroxidase activityin both LM and AP plants compared to untreated controls. Interms of (1–¹⁴C)IAA destruction, GA₃ + 0.01 mg 1^–1triacontanol caused appreciable auxin breakdown (40%) in LMtissue, with GA₃ + 0.1 mg 1^–1 triacontanol giving a 43%decrease compared to untreated controls. In AP tissue, 10 µMGA3 increased auxin destruction by 188% whereas 0.1 mg I^–1triacontanol caused a 20% decrease compared to untreated controls.The effects of triacontanol on root and stem growth, peroxidaseactivity, and auxin destruction appear to be cultivar-specific,with respect to LM and AP varieties of peas.     

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; )     

Auxin-regulated Wall Loosening and Sustained Growth in Elongation

It is proposed that auxin regulates and coordinates both wall loosening and the supply of wall materials in elongation. The tenets of the proposal allowed testable predictions. It was determined that, if the cell walls of Glycine max L. var. Wayne hypocotyl segments are maintained in a loosened state (by excising the segments directly into pH 4 medium), exogenous auxin induced only the second response. It was also predicted and confirmed that elongating systems, e.g. pea epicotyl, with certain early auxin-induced growth kinetics (an initial high non-steady-state rate followed immediately by a drop to a lower steady-state rate) would show a transient second response (in addition to the usual transient first response) when stimulated by pH 4 medium. Finally, it is pointed out that recent results which establish the existence of auxin-induced elongation-associated proteins support the proposition that auxin coordinates wall loosening and the supply of wall materials in elongation.     

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.     

Developmental and genotypic differences in the response of pea stem segments to auxin

The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; IAA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GC/MS, across genotypes and between early-expansion and mid-expansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbornadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been emonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin.     

A possible role of hydroxyproline-protein in oxygen-sensitive growth

Exposure of Avena coleoptile sections to 8% O₂ brought aboutrespiration decrease, resulting in a decrease of ATP production.The pH at the cell wall surface slightly rose in sections exposedto 8% O₂, while their growth was greatly accelerated. Moreover,this growth acceleration was observed even in sections treatedwith CCCP known to make membranes permeable for protons. Weconcluded that the growth acceleration with reduction of O₂concentration is probably not the result of secretion of H₊ions into cell wall compartments. Results of this study provided evidence to support the hypothesisthat there is an inverse relationship between hydroxyproline-proteinlevel and the ability of a cell to undergo rapid cell elongation.Total labeling of the cell wall fraction with ¹⁴C-proline wasunaffected by 8% O₂ treatment, although the radioactivitiesof hydroxyproline incorporated into this fraction during thetreatments fell to about 45% of the control. Moreover, the radioactivitiesof hydroxyproline incorporated into the SLS-insoluble cell wallfraction of sections exposed to 8% O₂ decreased to about 30%of the control. This decrease of hydroxyproline was also observedin sections treated with cycloheximide, which inhibits the secretionof H₊ ions into the cell wall compartment. Reduction of O₂ concentrationin the surrounding atmosphere affects not only the hydroxylationof peptidyl proline, but also the binding of hydroxyproline-protein(s)to cell wall polysaccharides, and the resulting decrease ofthe protein rigidly bound to them may induce cell elongation. (Received December 5, 1975; )     

Cell Wall Metabolism in Developing Strawberry Fruits

Cell wall metabolism was studied in strawberry receptacles (Fragariaananassa, Duchesne) of known age in relation to petal fall (PF).Polysaccharide and protein composition, incorporation of [¹⁴C]glucoseand [¹⁴C]proline by excised tissue, and the fate of ¹⁴CO₂ fixedby young, attached fruits were followed in relation to celldivision, cell expansion, fine structure, and ethylene synthesis. Cell division continued for about 7 d after PF although vacuolationof cells was already beginning at PF and the subsequent cellexpansion was logarithmic. There was an associated logarithmicincrease in sugar content per cell and a decreasing rate ofethylene production per unit fresh weight. During cell expansion radioactivity from [¹⁴C]glucose was incorporatedinto fractions identified as starch and soluble polyuronideand into glucose and galactose residues in the cell wall. Radioactivityfrom [¹⁴C]proline was also incorporated into the cell wall,but only 10 per cent of this activity was found in hydroxyproline.Correspondingly wall protein contained a low proportion of hydroxyprolineresidues. The proportion of radioactivity from ¹⁴CO₂ fixed byfruitlets remained constant in most sugar residues in the cellwall. The proportion of radioactivity in galactose fell, indicatingturnover of these residues. Between 21 and 28 d after PF receptacles became red and softenedbut there was no change in the rate of ethylene production.Cell expansion continued for at least 28 d. Tubular proliferationof the tonoplast and hydration of middle lamella and wall matrixmaterial had begun 7–14 d after PF but became extremeduring ripening. Associated with the hydration of the wall,over 70 per cent of the polyuronide in the wall became freelysoluble, and arabinose and galactose residues lost from thewall appeared in soluble fractions. There was no increase intotal polysaccharide during ripening and incorporation of [¹⁴C]glucoseinto polysaccharides ceased, although protein increased andincorporation of [¹⁴C]proline into wall protein continued.     

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.     

Cortical Cell Fluxes of Phosphate in Excised Root Segments of Allium cepa L.

Compartmental analysis was used to study content and transmembranefluxes of phosphate in excised root segments in a nutrient solutioncontaining 1.0 mol phosphate m^–3, at pH 5.6. The resultingradioactivity content versus time curve deviated from the criteriadetermining a relationship conforming to first order kinetics,since the final rate constant was an order of magnitude lowerthan that exhibited by the curve for efflux versus time. Thedata were manipulated so as to bring about conformation to theappropriate criteria, revealing a large, slowly exchanging poolthat is considered to represent assimilated phosphate or storedpolyphosphate. Subsequent calculations, using the modified data,suggested that H₂PO₄ was absorbed by a phosphate pump at theplasmalemma, but was actively extruded from the vacuole afterpassive entry. The consequences of pH effects in the cell wall,cytoplasm and vacuole in determining the concentration of H₂PO^–₄in each phase are discussed. Key words: Phosphate compartmentation, pH, efflux     

Effects of IAA on oxygen-sensitive growth and on hydroxyproline protein level in cell wall

Indole-3-asscetic acid (IAA) accelerated the incorporation ofradioactivity derived from ¹⁴C-proline into the SLS-insolublecell wall fraction only when the sections were exposed to lowoxygen concentrations. However, IAA showed no effect on theratio of hydroxyproline to proline incorporated into the SLS-insolublefraction in both 20% and 8% O₂-treated sections. The amountof hydroxyproline rigidly bound to the cell wall increased withincreasing IAA concentration in 8% O₂-treated sections, whilethat of the 20% O₂-treated ones decreased with IAA treatment. On the other hand, IAA increased the amount of ¹⁴C-labeled hydroxyprolineincorporated into the SLS-insoluble fraction of sections treatedwith cycloheximide, and their elongation was greatly inhibited. Based on the results that O₂ and IAA affect the auxin-inducedand the oxygen- sensitive growth differently, we suggest thatboth types of growth may antagonize each other in response tochanges in O₂ and IAA concentrations, resulting in balancedgrowth in the cell. (Received October 7, 1977; )