The Effect of High Concentrations of Auxin on the Extension of Avena Coleoptile Sections

The response of Avena coleoptile sections to high concentrationsof auxin has been determined in the absence of all additivesexcept sucrose. In most experiments the growth-time curves with75 p.p.m. IAA showed two linear phases. In the first phase,which lasted for only 2–4 hours, extension was as rapidwith 75 p.p.m. IAA as with 5 p.p.m. IAA. This rapid initialexpansion phase was then succeeded by a second phase which persistedfor at least 20 hours. During this second linear phase the growth-ratewith 75 p.p.m. IAA was lower than with an auxin concentrationof 5 p.p.m. In some experiments the first phase was absent andonly the second phase was present. The response of sections to high concentrations of auxin wasnot influenced by the presence of buffers or absorbable cations.Omission of sucrose or the presence of moderate amounts of ethanolcaused the resulting growth curves to be non-linear. The rate of uptake of auxin into the tissues was dependent onthe auxin concentration and was constant for at least 24 hours.     

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.     

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.     

Studies on Extension Growth in Coleoptile Sections: I. The Influence of Age of Coleoptile upon the Response of Sections of IAA

One of the problems involved in carrying out large-scale coleoptilecylinder straight growth assays is the effect of coleoptileage upon the behaviour of the sections. This effect, as measuredboth by the length of coleoptile and the time from sowing theseeds, has been investigated by an examination of the growthof sections cut from coleoptiles of several length grades onfive occasions 6–10 hours apart. The response of thesesections to a range of concentrations of ß-indolylaceticacid (IAA) was followed during the period of growth to ascertainthe most suitable time for measurement of sections in a routinetest. A number of general trends associated with coleoptile age (whethermeasured by length, or time from planting) were observed inamount of growth, growth-rate, period of growth, and sensitivity.It is suggested that these characteristics are associated withthe amount of the total growth already made by the parent coleoptileat the time of taking the section, and also that the sectionexhibits an ‘intrinsic growth-rate’ related to thegrowth-rate of the coleoptile at the time the section is cut. A period of growth of 17–20 hours seems satisfactory forroutine tests except where very young coleoptiles are used;in this case growth less than that in water is sometimes observedin very low concentrations of IAA.     

A Synergistic Stimulation of Avena sativa Coleoptile Elongation by Indoleacetic Acid and Carbon Dioxide

The ability of 0.03% CO₂ to stimulate growth has been investigated using etiolated Avena coleoptile sections maintained in buffered solution. This concentration of CO₂-stimulated growth after a lag period of 12 to 15 minutes, and a synergistic relationship between indoleacetic acid and CO₂ in stimulating growth has been demonstrated. The response to CO₂ is inhibited by cycloheximide and is lost approximately 10 minutes after exposure to CO₂-free air. Malate can replace CO₂ in stimulating growth. In the light of these data and recent literature on a growth response of coleoptile sections to CO₂-saturated solutions, the existence of two mechanisms of CO₂-simulated growth is proposed. In addition, it is suggested that growth promotion by 0.03% CO₂ is mediated by a process involving dark CO₂ fixation.     

The Extension Growth-Time Relationship for Avena Coleoptile Sections

1. The growth rates of coleoptile segments supplied with indole-3-aceticacid is not constant with time, but, when the IAA concentrationis high, decreases very rapidly.
2. With sufficiently high concentrationof IAA, the initial rapidgrowth may be eventually followedby shrinkage of the tissue.
3. The relation between initial rateof growth and auxin concentrationis not significantly differentfrom hyperbolic.
4. The significance of these facts in relationto kinetics of auxinaction is discussed.

     

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.     

Role of Auxin in Growth of Inhibitor-treated Oat Coleoptile Tissue

The role of auxin in the recovery of plant tissue from oxidant treatment was investigated. Treatment of oat coleoptile sections with concentrations of indoleacetic acid (IAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) optimal for normal growth, following pretreatment with moderately inhibiting levels of peroxyacetyl nitrate (PAN) immediately accelerated recovery of growth rate. In some cases inhibition was also less at supraoptimal values of auxin. Treatment of ozonepretreated tissue with IAA or 2,4-D enhanced inhibition at high levels of auxin and produced an optimal growth concentration level which was lower than for sections not given ozone pretreatment. Auxin treatment also reduced the degree of inhibition in fluoride and iodoacetamide-pretreated sections. Mechanisms by which auxin-induced recovery from inhibition may occur are discussed.     

Osmoregulation by Oat Coleoptile Protoplasts (Effect of Auxin)

The effect of auxin on the physiology of protoplasts from growing oat (Avena sativa L.) coleoptiles was investigated. Protoplasts, isolated iso-osmotically from peeled oat coleoptile segments, were found to swell steadily over many hours. Incubated in 1 mM CaCl2, 10 mM KCl, 10 mM 2-(morpholino)ethanesulfonic acid/1,3-bis-[tris(hydroxymethyl)methylamino]propane, pH 6.5, and mannitol to 300 milliosmolal, protoplasts swelled 28.9% [plus or minus] 2.0 (standard error) after 6 h. Addition of 10 [mu]M indoleacetic acid (IAA) increased swelling to 41.1% [plus or minus] 2.1 (standard error) after 6 h. Swelling (in the absence of IAA) was partially dependent on K+ in the bath medium, whereas auxin-induced swelling was entirely dependent on K+. Replacement of mannitol in the bath by Glc increased swelling (in the absence of IAA) and eliminated auxin-induced swelling. Swelling with or without IAA was inhibited by osmotic shock and was completely reversed by 0.1 mM NaN3. Sodium orthovanadate, applied at 0.5 mM, only gradually inhibited swelling under various conditions but was most effective with protoplasts prepared from tissue preincubated in vanadate. Our data are interpreted to suggest that IAA increases the conductance of the plasma membrane to K+.