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.     

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 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; )     

Turgor-dependent Changes in Avena Coleoptile Cell Wall Composition

The effects of reduced turgor pressure on growth, as measured by cell elongation, and on auxin-mediated changes in cell walls, as measured by analyses of wall composition, were examined using Avena coleoptile segments. Although moderate (1-4 bar) decreases in turgor resulted in a progressive decline in growth proportional to the decrease in turgor, the major auxin-induced change in wall composition, a decrease in noncellulosic wall glucose, was unaffected. Severe (5-8 bar) decreases, however, did inhibit this auxin effect on the wall, and with turgor decreases of 9 bars or more this auxin effect was no longer apparent. The results show that turgor pressure is required for this auxin-mediated wall modification and also that this modification of wall glucose occurs at turgor pressures less than those required for wall extension. Changes in other wall components were generally unaffected by altering turgor pressure.     

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.     

Auxin increases the hydraulic conductivity of auxin-sensitive hypocotyl tissue

The ability of water to enter the cells of growing hypocotyl tissue was determined in etiolated soybean (Glycine max (L.) Merr.) seedlings. Water uptake was restricted to that for cell enlargement, and the seedlings were kept intact insofar as possible. Tissue water potentials ( _w) were measured at thermodynamic equilibrium with an isopiestic thermocouple psychrometer. _wwas below the water potential of the environment by as much as 3.1 bars when the tissue was enlarging rapidly. However, _w was similar to the water potential of the environment when cell enlargement was not occurring. The low _w in enlarging tissue indicates that there was a low conductivity for water entering the cells.The ability of water to enter the enlarging cells was defined as the apparent hydraulic conductivity of the tissue (Lp). Despite the low Lp of growing cells, Lp decreased further as cell enlargement decreased when intact hypocotyl tissue was deprived of endogenous auxin (indole-3-acetic acid) by removal of the hypocotyl hook. Cell enlargement resumed and Lp increased when auxin was resupplied exogenously. The auxin-induced increase in Lp was correlated with the magnitude of the growth enhancement caused by auxin, and it was observed during the earliest phase of the growth response to auxin. The increase in Lp appeared to be caused by an increase in the hydraulic conductivity of the cell protoplasm, since other factors contributing to Lp remained constant. The rapidity of the response is consistent with a cellular site of action at the plasmalemma, although other sites are not precluded.Because the experiments involved only short times, auxin-induced changes in cell enlargement could not be attributed to changes in cell osmotic potentials. Neither could they be attributed to changes in turgor, which increased when the rate of enlargement decreased. Rather, auxin appeared to act by altering the extensibility of the cell walls and by simultaneously altering the ability of water to enter the growing cells under a given water potential gradient. The hydraulic conductivity and extensibility of the cell walls appeared to contribute about equally to the control of the growth rate of the hypocotyls.     

Changes in cell wall polysaccharides in the internodes of submerged floating rice

In excised stem segments of floating rice (Oryza sativa L.), as well as in intact plants, submergence greatly stimulates the elongation of internodes. The differences in the composition of cell wall polysaccharides along the highest internodes of submerged and air-grown stem segments were examined. The newly elongated parts of internodes that had been submerged for two days contained considerably less cellulosic and noncellulosic polysaccharides than air-grown internodes, an indication that the cell walls of the newly elongated parts of submerged internodes are extremely thin. In the young parts of both air-grown and submerged internodes, the relative amounts of noncellulosic polysaccharides were equal to those of -cellulose, whereas the relative amounts of -cellulose were higher than those of noncellulosic polysaccharides in the upper, old parts. In the cell-elongation zones of both air-grown and submerged internodes, glucose was predominant among the noncellulosic neutral sugars of cell wall. The relative amount of glucose in noncellulosic neutral sugars decreased toward the upper, old parts of internodes, whereas that of xylose increased.     

The specificity of the auxin transport system

Summary In an effort to examine the specificity of the auxin transport system, the movement of a variety of growth substances and of auxin analogues through corn coleoptile sections was measured in both the basipetal and acropetal directions. In contrast to the basipetal, polar transport of the auxins indoleacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid, no such movement was found for benzoic acid or for gibberellin A₁. A comparison of the - and -isomers of naphthaleneacetic acid showed that the growth-active -form is transported, but not the inactive -analogue. Both the dextro (+) and leavo (-) isomer of 3-indole-2-methylacetic acid showed the basipetal movement characteristic of IAA, the dextro isomer being more readily transported than the (-)-form. In this instance, too, the transport was roughtly proportional to the growth promoting activity. The antiauxin p-chlorophenoxyisobutyric acid inhibited auxin transport as it inhibited auxin-induced growth. These results agree with the hypothesis that processes involved in auxin transport are closely linked to or even identical with the primary auxin action.     

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.     

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; )     

Inhibition of Golgi-apparatus function by brefeldin A in maize coleoptiles and its consequences on auxin-mediated growth,cell-wall extensibility and secretion of cell-wall proteins

Brefeldin A (BFA), a fungal metabolite causing dysfunction of the Golgi apparatus in plant and animal cells, was used to investigate the role of secretory processes at the plasma membrane in auxin-mediated elongation growth of maize (Zea mays L.) coleoptiles. In abraded coleoptile segments BFA produced, within less than 30 min, a decrease in the incorporation of [³H]leucine into tightly bound cell-wall proteins, accompanied by an increased incorporation into the intracellular pool of putative cell-wall glycoproteins. Total protein synthesis was not affected. Electron micrographs revealed striking morphological changes in dictyosomes (especially vesiculation of trans-cisternae), accumulation of Golgi vesicles and dilation of the endoplasmic reticulum. These effects are taken as indication that BFA interferes with the secretion of cell-wall components. Elongation growth of coleoptile segments in the presence and absence of auxin was inhibited by 80% in 20 mg·l^–1 BFA. If BFA was applied to segments growing in the presence of auxin, maximum inhibition was reached after about 30 min, indicating that the growth response depends on an uninterrupted supply of a cell-wall or plasma-membrane component (wall-loosening factor) delivered by the secretory pathway. After its secretion, this factor has a rather short growth-effective life time. The inhibition of auxin-mediated growth by BFA was accompanied by an elimination of auxin-induced cell-wall extensibility and by an inhibition of auxin-induced proton excretion. Fusicoccin-induced proton excretion was similarly affected by BFA. It is concluded that both the wall-loosening process underlying elongation growth as well as proton excretion depend on an intact secretory pathway from the Golgi apparatus to the cell wall; however, a causal relationship between these processes is not warranted by the data.Abbreviations BFA brefeldin A - FC fusicoccin - TCA trichloroacetic acid - WLF wall-loosening factor Supported by Deutsche Forschungsgemeinschaft (SFB 206). We thank Ms. B. Huvermann and Mrs. C. Plachy for conducting growth and proton excretion measurements.     

Effect of auxins on the auxin transport system in coleoptiles

Summary Within 20 min of its application, auxin enhances the transport processes that move auxin molecules through coleoptile tissue. The effect was observed after treatment with indoleacetic acid and -naphthaleneacetic acid, but not with growth-inactive auxin analogues.     

Concanavalin A Inhibits Auxin-Induced Elongation and Breakdown of (1 -> 3), (1 -> 4)-{beta}-D-Glucans in Segments of Rice Coleoptiles

Concanavalin A (Con A) suppresses auxin-induced elongation ofsurface-abraded segments from both dicotyledonous and poaceousplants. In coleoptile segments of rice (Oryza sativa L.), theauxin-induced decrease in the minimum stress-relaxation timeand increase in the mechanical extensibility of the cell wallswere also inhibited by Con A, indicating that the lectin suppresseselongation by inhibiting the cell wall loosening. Auxin causeda decrease in the level of (1 3), (1 4)-ß-D-glucansin the cell walls of rice coleoptile segments, and this decreasewas also inhibited by the lectin. Con A suppressed the autolytichydrolysis of the glucans, as well as their breakdown in vitroby a protein fraction that had been extracted from the cellwalls of rice coleoptiles with 1 M NaCl. Furthermore, most ofthe glucan-hydrolyzing activity of the wall proteins bound toa Con A-Sepharose column, suggesting that glycoprotein enzymesare involved in the hydrolysis. Although Con A also affectedthe hydrolysis of other wall polysaccharides, the present data,when considered in combination with the inhibitory effects ofglucan-specific or glucanasespecific antibodies, support theview that the breakdown of (1 3),(1 4)-ß-D-glucansis associated with the cell wall loosening that is responsiblefor auxin-induced elongation in Poaceae. (Received August 17, 1994; Accepted February 15, 1995)     

Cytological studies on the inhibition by 5-fluorouracil of ribosome synthesis and growth in jerusalem artichoke tuber slices

Summary Rapid auxin-induced cell expansion in artichoke tuber slices is obtained by aerating the slices in water (aging) prior to auxin treatment. 5-fluorouracil (5-FU), an inhibitor of ribosomal RNA synthesis in plant cells, markedly inhibits auxin-induced growth only if present in the pre-growth aging period. Autoradiographic studies show that 5-FU given in the aging and/or growth periods reduces the incorporation of RNA precursors into the cytoplasm. Pulse-chase experiments suggest that the reduced cytoplasmic incorporation is in large part due to decreased stability of ribosomal rNA, as nucleolar and chromatin label are only slightly depressed at the end of the pulse. Though the nucleoli continue to incorporate RNA precursors following 5-FU treatment, they lack a distinct granular zone, and appear as homogeneous fibrillar structures under the electron microscope. 5-FU has a parallel inhibitory effect on growth and protein synthesis as shown by ³H-leucine studies during the growth period. Electron-microscope studies show that treatment with 5-FU causes decreased numbers of ribosomes and rough endoplasmic reticulum. The results suggest that the ribosomes and rough endoplasmic reticulum formed during aging are important in obtaining subsequent rapid auxin-induced expansion. The new ribosomes serve in part to replace pre-existing ribosomes present at the time of excision, which from electron microscopic evidence from 5-FU treated tissue, appear to slowly disappear.     

Auxin-induced cell expansion in relation to cell wall extensibility

Decapitation of 30 mm oat coleoptiles, which are commonly usedfor growth tests, resulted in a decrease in their elastic extensibility(DE) but not in their plastic extensibility (DP). By auxin treatmentunder osmotic stress, old coleoptile (45 mm) cells showed noincrease in subsequent expansion in water, whereas RNA synthesisin these cells was stimulated just as in young ones. Auxin increasedthe DE of young coleoptile cell walls but not that of old ones.Significant increase of DE occurred in only 10 min, and themaximum level of DE was reached in 15 min of the auxin treatment.An antiauxin (2,4,6-trichlorophenoxyacetic acid), mitomycinC and cycloheximide inhibited auxin-induced increases in expansionand DE (or Rex, reversible extensibility) of young coleoptilecells. (Received July 23, 1968; )     

Phytochrome in etiolated annual rye

Summary During a seven-fold increase in length the content of the coleoptile in photoreversible phytochrome increased four-fold and that of the primary leaf nine-fold. The phytochrome content, during growth, expressed on a fresh- or dry-weight basis did not vary greatly for either organ. Phytochrome per mg dry weight (OD⁷³⁰/mg=0.5) was nearly the same in the leaf as in the coleoptile. Coleoptiles studied had a constant DNA content of 4.1 g per organ. DNA content of the leaf increased with age. Phytochrome per DNA was much higher in the coleoptile than in the primary leaf and increased with growth in each of these organs. Thus, there was not a constant amount of phytochrome per cell in either tissue with increasing age and there was not the same amount of phytochrome per cell in the coleoptile as in the primary leaf at any age.This work was supported in part by U.S. Atomic Energy Commission Contract No. AT (30-I)2373.     

The “acid growth effect” and geotropism

Summary The curvature developed by segments of sunflower hypocotyl exposed to gravitational stimulus was enhanced in buffer solutions between pH 3.4 and 4.0 in the absence of added auxin. This effect was observed both when the segments were submerged during the stimulus and when they floated near the surface of the solution. 5–10 min in a horizontal position was sufficient to induce subsequent curvature.Straight growth of the segments was also promoted in buffers of this pH range.The acid effect on curvature was insensitive to KAsO₂, HgCl₂ and cycloheximide, inhibitors which drastically reduced auxin-induced curvature. Furthermore, acid buffer, but not auxin, restored the ability of segments taken from etiolated and starved plants to respond to gravity. These results suggest that the polarisation following gravistimulus may not be resticted to the asymmetric distribution of auxin and auxin co-factors but may involve a general physiological asymmetry.     

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.     

Continuous measurement of initial curvature of maize coleoptiles induced by lateral auxin application

To analyze early effects of auxin application, an apparatus was developed which continuously and simultaneously registered the curvature of 10 individual maize (Zea mays L.) coleoptiles. Resolution was less than 5 m over a range of ±0.5 mm. The data were evaluated and plotted via paper tape and Hewlett-Packard-computer. Unilateral application of 3×10^-5 M indoleacetic acid (IAA) resulted in a transient inhibition of growth on the side of application for ca. 10 min (Phase I), followed by a strong stimulation (Phase II). The phytotoxin fusicoccin (FC) caused an immediate stimulation of elongation. The initial negative reaction of Phase I is auxin-specific. Only active auxins such as IAA and 1-naphtaleneacetic acid produced this initial inhibition; chemical analogs-inhibitory or neutral in long-term growth tests, e.g. phenylacetic acid-did not show any significant effects on Phase I. When the coleoptiles were symmetrically preloaded with different levels of auxin, only a large step-up of subsequent unilateral auxin application resulted in a negative phase I; a small step-up led to an immediate positive reaction. The results are discussed in context with the parallel kinetics for various other auxin-induced reactions of coleoptile cells which have already been published.Abbreviations FC fusicoccin - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - PAA phenylacetic acid     

Rapid-growth responses of corn root segments: Effect of auxin on elongation

The effect of indole-3-acetic acid (IAA) on the elongation rates of 2 mm corn (Zea mays L.) root segments induced by citrate-phosphate buffer (or unbuffered) solutions of pH 4.0 and 7.0 was studied. At pH 7.0, auxin initially reduced the elongation rate in both buffered and unbuffered solutions. Only in buffer at pH 7.0 was auxin at a concentration of 0.1 M found to promote the elongation rate though briefly. THis promoted rate represented only ca. 20% of the rate achieved with only buffer at pH 4.0. Auxin in pH 4.0 buffered and unbuffered solutions only served to reduce the elongation rates of root segments. Some comparative experiments were done using 2 mm corn coleoptile segments. Auxin (pH 6.8) promoted the elongation rate of coleoptile segments to a level equal or greater than the maximal H ion-induced rate. The two responses of root segments to auxin are compared to auxin action in coleoptile growth.