The East Malling Coleoptile Straight Growth Test Method

The very simple coleoptile straight growth test used for manyyears at East Malling is described, and various modificationsmentioned. Experiments are described on which some of the points of techniquewere based, such as rates of rotation, size of tube, volumeof solution, &c.     

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.     

Changes in the Autolytic Activities of Maize Coleoptile Cell Walls during Coleoptile Growth

Autolytic activities of coleoptile cell walls were measuredin developing maize seedlings. The major neutral sugar componentsof the cell wall polysaccharides were arabinose, xylose andglucose. The quantities of all these components per coleoptileincreased for 5 d after germination, suggesting that levelsare augmented by biosynthetic processes during coleoptile growth.However, cell wall preparations isolated from the coleoptilesalso revealed increasing rates of autolytic activity directedtoward each of the sugar components. This result suggests thatthe constitutive hydrolytic activities expressed by cell wallsalso increase as a function of coleoptile age. The proportionof glucose in autolysis products relative to that present inthe cell walls specifically increased with coleoptile age, whilethe ratios for arabinose and xylose decreased. Kinetic analysesof autolysis demonstrated that the reactions specific for pentosesat the early growth stage are transient events and that initiallow rates of glucan autolysis increased sharply and persistedlonger. In these experiments the products of glucan autolysiswere largely monomeric while those of the pentose-specific reactionsconsisted of both monomeric and polymeric sugars. Based on theseresults, we concluded that two distinct phases of autolyticactivities are expressed in the mediation of cell wall polysaccharidemetabolism in situ. (Received July 17, 1996; Accepted November 25, 1996)     

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.     

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.

     

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.

     

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.     

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.     

The Effect of Gibberellin on Tryptophan Conversion and Elongation of the Avena Coleoptile

Gibberellic acid (GA₃) enhanced directly the release of ¹⁴CO₂ from tryptophan-1-^l4C by cell free preparations of Avena coleoptile tips. The rate of tryptophan metabolism in the presence of GA₃ was increased by approximately 100 per cent. The addition of auxin synthesis inhibitors to incubation flasks nullified the enhancement effect of GA₃ on elongation of the coleoptile tips. These studies implicate tryptamine as an intermediate in the formation of auxin from tryptophan. The possibility of GA₃-IAA interaction in the elongation processes was also investigated. Combination treatments of these growth-promoting substances did not induce a synergistic growth response by the coleoptile tissue.     

Auxin-stimulated RNA Synthesis in Oat Coleoptile Cells

Using oat coleoptile segments the following results were obtained. Ten mg/l auxin (indole-3-acetic acid) increased the incorporation of uracil-2-¹⁴C and orthophosphate-³²P into RNA fraction during a relatively short incubation period. Stimulation of ³²P incorporation due to auxin was found only in the region heavier than ribosomal RNA, probably in the messenger RNA region. The stimulation of uracil-2-¹⁴C incorporation into RNA caused by auxin was not influenced by the presence of 0.3 M mannitol which prevents osmotically the water absorption of cells. It is concluded that auxin primarily stimulates the biosynthesis of RNA, possibly messenger, in oat coleoptile cells.     

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.     

Rapid Hormone-induced Hyperpolarization of the Oat Coleoptile Transmembrane Potential

The effects of the plant growth substances indoleacetic acid (IAA) and fusicoccin on the transmembrane potential of Avena coleoptile cells (at 27-29 C) were studied. Fusicoccin caused hyperpolarization of the membrane potential which started after a lag of less than 20 seconds, and which on average reached -49 mv at an external K(+) concentration of 1 mm and -75 mv at 0.1 mm K(+). IAA caused a hyperpolarization of -25 mv starting after a lag of 7 to 8 minutes. These results suggest that fusicoccin and IAA both activate electrogenic H(+) extrusion.