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.     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

Culturing Puccinia coronata on a Cell Monolayer of the Avena sativa Coleoptile

A culture of Puccinia coronata on an epidermal cell monolayer dissected from the Avena sativa caleoptile is described. To induce fungal development, infection structure formation had to be induced with a heat treatment. The host cells were fed with 1 % sucrose (or glucose or manitol) to sustain fungal growth. Under these conditions, normal development of hyphae, haustoria with haustorial mother cells and uredospores occurred. Uredospores had normal infectivity.     

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.

     

Inhibitory Effects of Dichlorophenoxyacetones on Auxin-induced Growth of Avena Coleoptile Sections

Six dichlorophenoxyacetones were synthesized and examined as potential metabolic antagonists utilizing Avena coleoptile sections and the straight growth assay procedure. Supplements of indoleacetic acid promoted growth of the sections which were inhibited by the analogs; the most inhibitory derivatives were 2,3-; 2,4-; 2,5-; and 3,4-dichlorophenoxyacetone which produced half-maximal growth responses (relative to the unaug-mented control growth) at concentrations of 106, 86, 80, and 62 μg/ml, respectively. A Lineweaver-Burk plot of the data for the inhibition by 2,4-dichlorophenoxyacetone and its reversal by indoleacetic acid appeared to represent an uncompetitive-like inhibition.     

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.     

Rapid Inhibition of Auxin-induced Elongation of Avena Coleoptile Segments by Cordycepin

The effects of cordycepin (3'-deoxyadenosine), an RNA synthesis inhibitor, on auxin-induced elongation in Avena coleoptile segments were studied with a position-sensing transducer. Cordycepin rapidly inhibited auxin-stimulated growth in the coleoptile segments whether added before, at the same time as, or after, the 2 mum auxin treatment. Midcourse additions of 100, 50, and 25 mug/ml cordycepin inhibited auxin-promoted elongation in an average of 18, 22, and 35 minutes, respectively. Additions of cordycepin before or at the same time as the auxin treatment partially inhibited the magnitude of the subsequent auxin-promoted growth but did not appreciably alter the latent period of the auxin response. It was concluded that if cordycepin is inhibiting the synthesis of RNA required for growth, the decay time for this RNA may be considerably shorter than that suggested in the literature from actinomycin D experiments. Preliminary kinetic evidence indicated that cordycepin does not inhibit auxin-induced elongation by acting as a respiratory inhibitor. Studies in mung bean shoot mitochondria demonstrated that cordycepin has no effect on respiration, respiratory control, or ADP/oxygen ratios.     

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.