Effect of pH and Auxin on Chloride Uptake into Avena Coleoptile Cells

The effect of pH on ³⁶Cl⁻ movement into coleoptile cells (Avena sativa L. cv. Garry) was investigated and compared with effects of indoleacetic acid. ³⁶Cl⁻ uptake, but not efflux, is stimulated when coleoptile sections are placed in media adjusted to pH levels from 5 to 3 after a preincubation period at pH 6.5. The enhancement is seen within 2 minutes, is not correlated with growth, and is completely erased by respiratory inhibitors. In comparison to the acid-induced stimulation, the stimulatory effect of indoleacetic acid on ³⁶Cl⁻ uptake is also not accompanied by accelerated efflux, and indoleacetic acid does not further stimulate ³⁶Cl⁻ uptake into 1-millimeter sections beyond that seen at pH 3.5 without auxin.     

Auxin Balance in the Avena Coleoptile

Coleoptiles of Avena possessed the capacity to degrade infiltrated indole-3-acetic acid (IAA). This activity decreased along the length of the coleoptile from apex to base on the bases of fresh weight, dry weight and protein; the apical 1 cm segment degraded more IAA than segments from other parts of the coleoptile. The naturally occurring inhibitor of the IAA oxidase activity increased in concentration up to 20 mm from the coleoptile apex; beyond, it decreased gradually towards the base. The spatial distribution of this inhibitor does not explain the gradient in IAA oxidase activity. Growth in length of the coleoptile and the IAA inactivating capacity of the apical 1 cm segment, increased 5- and 4,4-fold, respectively, between the ages of 70 and 130 h; but auxin secretion into agar platelets by the apical 2 mm of the coleoptile registered only a 2.7-fold increase. Deseeding and derooting the seedlings reduced the subsequent growth, diffusible auxin content and the IAA oxidase activity of the coleoptiles; derooting proved to be more deleterious than deseeding. A parallel reduction was evident in auxin content and IAA degrading activity following these treatments. Application of the cytokinin 6-benzylaminopurine (BAP) to coleoptiles of derooted seedlings failed to influence their capacity to degrade IAA. Nor was the activity of the aldehyde oxidase, which converts indole-3-acetaldehyde (IAAld) to IAA, affected by such treatment.     

beta-d-Glucan of Avena Coleoptile Cell Walls

A specific glucanase was used to liberate a noncellulosic beta-d-glucan from isolated cell walls of Avena sativa coleoptile tissue. Cell walls of this tissue contain as much as 7 to 9 mg of glucan/100 mg of dry wall. Because of the specific action pattern of the enzyme, a linkage sequence of.. 1 --> 4 Glc 1 --> 3 Glc 1 --> 4 Glc.. is indicated and the predominance of trisaccharide and tetrasaccharide as hydrolytic products suggests a rather regular repeating pattern in the polysaccharide. The trisaccharide and the tetrasaccharide are tentatively identified as 3-O-beta-cellobiosyl-d-glucose and 3-O-beta-cellotriosyl-d-glucose, respectively. Recovery of these oligosaccharides following glucanase treatment of native wall material was feasible only after wall-bound glucosidases were inactivated. In the absence of enzyme inactivation the released fragments were recovered as glucose. The beta-d-glucan was not extracted from walls by either hot water or protease treatment.Cell walls prepared from auxin-treated Avena coleoptile segments yielded less glucan than did segments incubated in buffer suggesting an auxin effect on the quantity of this wall component. No IAA-induced change in the ratio of the trisaccharide and tetrasaccharide could be detected, suggesting no shift in the 1,3 to 1,4 linkage ratio. While the enzyme acts directly on the beta-d-glucan, no elongation response was apparent when Avena sections were treated with the purified glucanase. The presence of the glucan was not associated with any wound response which could be attributed to the preparation of coleoptile segments. The relationship of glucan metabolism to auxin growth responses is discussed.     

The Intercellular Spaces of the Avena Coleoptile

In the transverse sections of fresh Avena coleoptile certain intercellular spaces are transparent, others are dark. The transparent spaces represent the result of water-logging of the originally water-lined air passages. The dark spaces are lined with a plastic lipid-containing membrane which can be impregnated with melted paraffin. In the living tissue this membrane can be cut transversely and the cut sections presumably seal off the gas inside thus causing the dark interfacial refraction. Because of the high permeability of lipids to carbon dioxide and the virtual impermeability to oxygen and nitrogen, there is a reason to believe that the lipid-lined spaces are filled with gas rich in carbon dioxide, and the lipid membrane may function as a regulator of the diffusion pressure of this gas.     

Auxin-induced Changes in Avena Coleoptile Cell Wall Composition

Sugar and uronic acid residues were derived from wall polysaccharides of oat (Avena sativa, var. Victory) coleoptiles by means of 2 N trifluoroacetic acid, 72% sulfuric acid, or enzymic hydrolysis. The products of hydrolysis were reduced and acetylated to form alditol acetates which were analyzed using gas chromatography. Time-course studies of auxin-promoted changes in various wall fractions indicate that when exogenous glucose was available, increases in certain wall constituents paralleled increases in length. However, under conditions where exogenous glucose was not available, and where wall synthesis was limited, such correlations with growth were not apparent. Under these latter conditions total wall weight initially increased slightly, then decreased. These changes in weight were the net of increases in cellulose and some noncellulosic constituents and a decrease of over 75% in noncellulosic glucose. When coleoptile sections were preincubated without exogenous glucose for 8 hours to deplete endogenous wall precursors and subsequently treated with auxin, there were no detectable increases in wall weight. There was instead an auxin-promoted decrease in wall weight, and this decrease paralleled a decrease in noncellulosic glucose. There were no significant changes in other wall components. The auxin-promoted decreases in noncellulosic glucose are interpreted as a possible step in the mechanism of growth.     

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.     

Inhibition of Cell Elongation in Avena Coleoptile by Hydroxyproline

A study has been made of the hydroxyproline-induced inhibition of elongation of Avena coleoptile tissues. The isomers of 4-hydroxyproline differ in their effectiveness; only the L isomers are growth inhibitors with the cis form (allohydroxyproline) being more effective than the trans form (hydroxyproline).Hydroxyproline differs from other amino acid antagonists and protein synthesis inhibitors in respect to 2 characteristics of the growth inhibition. First, a certain increment of auxin-induced elongation must take place following addition of hydroxyproline before the growth is inhibited. In contrast, pretreatment with other amino acid antagonists or protein synthesis inhibitors completely eliminates the ability of Avena coleoptile sections to respond to auxin. Secondly, sucrose markedly increases the magnitude of the hydroxyproline inhibition; i.e., sucrose acts to inhibit rather than promote growth when in the presence of hydroxyproline.It appears that hydroxyproline is a specific inhibitor for the synthesis of some factor which is utilized in elongation. Following addition of hydroxyproline, auxin-induced elongation continues until the pool of this factor is exhausted; then elongation is inhibited.     

Cyanide Inhibition of Acid-induced Growth in Avena Coleoptile Segments

The comparative effects of metabolic inhibitors on acid- and auxininduced growth in oat (Avena sativa L. var. Victory) coleoptile segments have been examined. Acid (pH 4)-induced growth in both peeled and unpeeled segments is inhibited by 1 millimolar KCN when added at the time of acidification. KCN inhibits total acid-induced growth by 59 and 76%, respectively, in peeled and nonpeeled segments during the first 60 minutes. The growth rate of cyanide-treated tissue drops to zero or near zero in both peeled and nonpeeled segments during this period. Cyanide inhibition of total acid-induced growth in peeled segments at pH 5 is even more severe, amounting to about 80% during the first 60 minutes. The possibility that inhibition by cyanide may be caused by some nonspecific effect of the inhibitor on a process other than respiration, e.g. turgor reduction due to membrane damage, has not been ruled out. Acid-induced growth is also inhibited by 3 millimolar sodium fluoride and by anoxia. In unpeeled segments total pH 4-induced growth is inhibited 73% by sodium fluoride and 38% by anoxia during the 1st hour. Possible corrections to the above inhibition percentages which may be necessary due to the sensitivity of basal growth to inhibitors are discussed. Cyanide was found to inhibit auxin-induced growth much more rapidly than acid-induced growth. These data suggest that acid growth may be dependent on respiratory metabolism but to a lesser degree than is auxin-induced growth. If the acid growth theory of auxin action is correct, it appears that there may be two steps in the growth process which are dependent on respiratory metabolism: (a) auxin-induced proton pumping which is highly sensitive to respiratory inhibitors; and (b) acid-mediated wall loosening which is moderately and perhaps indirectly sensitive to respiratory inhibitors.     

Stress-relaxation Properties of the Avena Coleoptile Cell Wall

Changes in the cell wall properties of Avena coleoptile segments were studied under various conditions by stress-relaxation analysis. Rheological models consisting of four or an infinite number of Maxwell viscoelastic components were used. The stress-relaxation parameters of these models, t₁, t_o, T, Gi and stress/strain ratio, were determined. The following results were obtained. 1. The 1/T₁ increased and stress/strain ratio decreased with the age of the coleoptiles. Decapitation caused a decrease in l/t₁. 2. Auxin increased I/T₁ but decreased t_o and stress/strain ratio within 5 minutes after application. 3. Treatment with a fungal β-l,3-glucanase increased 1/T₁ both in living and methanol-killed, pronase-treated coleoptiles. Cellulase did not cause the changes observed in the parameters of the isolated cell wall of the coleoptile segments. This held true for all treatments (with and without auxin, killed and pronase-treated). The results obtained suggest that auxin primarily causes a partial degradation of the non-cellulosic physaccharide components of the cell wall.     

Investigations of the Geotropic Curvature of the Avena Coleoptile

The geotropic reaction in Avena coleoptiles is studied as a function of the stimulation time. The direction of the stimulation with respect to the vascular bundles must be defined when studying geotropic responses. It is found that the threshold time to evoke geotropic response is less than half a minute, i.e., at least ten times lower than the presentation time usually reported in the literature. An extrapolation procedure can be used to give a so-called extrapolated presentation time t_b, which is intimately related to the logarithmic part of the geotropic response curve and has a physical meaning in the reciprocity rule. The problem of the duration of the true threshold time for stimulation with 1 g is discussed. An experiment indicates that it is not necessary for mass particles (“statoliths”) to settle on the lateral cell wall in order to start the geotropic reaction chain. The slope of the logarithmic part of the geotropic response curve is independent of the transverse force applied to the coleoptiles. Support is given to the view that the slope is determined by the number of sedimenting mass particles.     

Direct and Synergistic Influence of Tweens on Avena Coleoptile Section Elongation

The Avena cylinder test was used with Tweens at 3 different concentrations with and without the addition of varying concentrations of IAA. Tween 20 was a toxic substance for oat coleoptile sections at such concentration as 0.1 %. Tween 40 gave erratic results and Tween 80 has practically no direct effect on cell elongation. An inhibitory effect continues to increase with increasing HLB values. The second object of this experiment was to ascertain whether cell elongation promoted by IAA would be enhanced by osmotic substances such as Tweens, as consequence of a specific modification in the permeability when the membranes are lipid in nature. The hypothesis was supported by the experimental work when 0.05 % Tweens were assayed although the inverse was also true, with concentrations of Tweens as low as 0.01 %. Accordingly, evidence must be verified by further investigation.