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.     

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.     

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.     

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.     

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.     

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.     

Cell Physiological Studies on the Avena Coleoptile Treated with Ribonuclease

It was revealed with excised Avena coleoptile that the growth promoting effect of indole-3-acetic acid was inhibited by pretreatment with ribonuclease (Masuda 1959a, b). This effect of ribonuclease was presumed to involve its digestive action on the ribonucleic acid at the protoplasmic surface (Masuda 1959b). Ribonuclease treatment decreases the cation binding capacity of the ribonucleic acid at the protoplasmic surface (Masuda 1959a).
On the other hand, it has been confirmed that indole-3-acetic acid bas a remarkable effect on the physico-chemical properties of protoplasmic surface such as permeability (Masuda 1955) and adhesiveness of protoplasm to the cell wall (Masuda 1957, Masuda and Takada 1957).
The purpose of the present study is to see the effect of ribonuclease on some protoplasmic properties of cells of Avena coleoptile and substantiate the authors view on the participation of ribonucleic acid in the cell elongation.     

Osmoregulation in the Avena coleoptile in relation to auxin and growth

A study has been made of the effects of auxin and growth on the ability of Avena coleoptile sections to osmoregulate, i.e. to take up solutes so as to maintain their osmotic concentration, turgor pressure, and growth rate. The high auxin-induced growth rate of Avena coleoptiles is maintained when cells are provided sucrose, glucose, NaCl, or KCl as a source of absorbable solutes, but not when 2-deoxy-d-glucose or 3-O-methyl-d-glucose is used. In the absence of auxin, cells take up solutes from a 2% sucrose solution and the osmotic concentration increases. The rate of solute uptake is even greater in the presence of auxin or fusicoccin, but the osmotic concentration rises only slightly because of the water taken up during growth. Solute uptake is not stimulated by auxin when growth is inhibited osmotically or by calcium ions. Solute uptake appears to have two components: a basal rate, independent of auxin or growth, and an additional uptake which is proportional to growth. Osmoregulation of sections may be limited by the rate of entry of solutes into the tissue rather than by their rate of uptake into the cells.     

The Growth-time Relationships of the Auxin- induced Growth in Avena Coleoptile Sections

1. 1. The growth rate of Avena coleoptile sections in the presenceof indoleacetic acid (IAA) is constant with time over a widerange of time intervals and IAA concentrations.
2. 2. Constancyof growth rate is dependent upon the maintenanceof constantconditions in which the concentration of IAA availableto thesection remains the chief factor limiting growth rate.
3. 3.Control of the pH of the medium in which the sections aregrownis essential to the maintenance of constant growth rate,particularlyin the presence of high concentrations of IAA.
4. 4. The lagperiod in establishment of steady growth rate bysections inthe presence of IAA is less than 10 minutes andis not detectableby present methods of measurement.

     

An Endogenous 24-hour Rhythm in the Growth Rate of the Avena Coleoptile

The rates of growth of coleoptiles of intact Avena seedlingswere studied by means of time-lapse photography, using infra-redradiation. When the seedlings are germinated in red light and subsequentlytransferred to darkness, a growth rhythm is established in whichthe first peak in the growth-rate curve occurs about 16–17hours after the transfer, and the second peak 24 hours later.When the transfer is made sufficiently early, three peaks mayoccur before growth ceases. The occurrence of the peaks andthe emergence of the primary leaf are independent of one another. Alteration of the point in the life-history at which the seedlingsare transferred from light to darkness changes the times ofoccurrence of the peaks, but does not affect the period of therhythm. The incidence of the rhythm shows no correlation withtime of day; therefore the rhythm is not due to diurnal changesin external conditions. Interruption of the dark period by several hours' exposure tored light causes the suppression of a previously induced rhythmand the establishment of a new one which commences at the timethe seedlings are restored to darkness. When they are grownunder continuous red light no rhythm is induced. Within the range 16 to 28 C., temperature has little or noeffect on the period of the rhythm. When seedlings of Triticum are grown under the same conditionsas those which induce a rhythm in Avena, no rhythmical variationin the growth rate can be detected.     

Antagonism Between Malate and Ethylene in the Regulation of Avena sativa Coleoptile Elongation

Etiolated Avena sativa L. coleoptile sections were used to determinethe influence of C₂H₄ on in vivo and in vitro rates of CO₂ fixation,and to measure the influence of various permutations of C₂H₄,CO₂, and malate on growth. Whereas 1 mM malate or 320 µI^-1 CO₂ stimulated growth by approximately 100 per cent, inhibitionof growth by 10-8 µ I_-1 C₂H₄ was substantial only in thepresence of malate or CO₂ The increase in growth rate in responseto these two agents was eliminated by the simultaneous applicationof C₂H₄. The in vivo rate of dark [¹⁴C]bicarbonate fixationand in vitro enzymic assays of fixation were not measurablyinhibited by C₂H₄. These results are discussed in the lightof evidence which indicates that CO₂-stimulated growth is mediatedby dark fixation. The data do not support the view that C₂H₄inhibition of growth results from an inhibition of fixation,but suggests that C₂H₄ may inhibit some step in the processby which malate stimulates growth.     

Osmoregulation in Rice Coleoptile Elongation as Promoted by Cooperation between IAA and Ethylene

IAA-induced elongation of rice (Oryza sativa L. cv. Sasanishiki)coleoptiles is regulated by cooperation between IAA and ethyleneproduced in response to IAA. However, the presence of some solutes,such as K^$, Na^$, Rb^$, glucose and sucrose, in the incubationmedia was found to be indispensable for this cooperation. Withoutthose solutes, the IAA-induced elongation was not sustainedover a long time period. IAA caused increases in both the osmoticpotentials of the coleoptile cells and the extensibility oftheir cell wall. In epidermal cells of IAA-treated coleoptiles,the osmotic potential increased from –0.87 to –0.62MPa during a 4-h incubation with 1 mM KCl. Moreover, IAA promotedthe uptake of K^$ or Na^$ from the media into the coleoptiles.However, these effects of IAA were partially prevented by aminoethoxyvinylglycine(AVG), and all the AVG effects were completely nullified byethylene applied simultaneously and exogenously. Both IAA andethylene did not affect the wall yield stress. These resultssuggested that the long-term elongation induced by IAA in ricecoleoptile segments results from inhibiting increases in osmoticpotentials of their cells. The maintenance by IAA of low osmoticpotentials may be partly due to the promotive action of ethyleneproduced in response to IAA on the solute uptake from the media. (Received July 6, 1983; Accepted February 15, 1984)