Coleoptile Tip Research Articles

The Dark Reactions of Rye Phytochrome in Vivo and in Vitro

The dark reactions of Secale cereale L. cv. Balbo phytochrome have been investigated in coleoptile tips and in extensively purified extracts of large molecular weight phytochrome. Destruction, but not reversion, was detected in vivo. The effects of various inhibitors of an in vitro phytochrome-degrading protease did not support a view of proteolytic attack as the basis of in vivo destruction. In vitro, rye phytochrome (about 240,000 molecular weight) reverted extremely rapidly, even at 5 C. The reversion curves were resolved into two first order components. The previously studied 60,000 molecular weight species, obtained by controlled proteolysis of large rye phytochrome, showed a similar two-component pattern, but a much slower over-all reversion rate. This reduction in rate was caused mainly by the reversion of a greater percentage of the small phytochrome as the slow component. Sodium dithionite markedly accelerated the reversion rate of both large and small forms, but oxidants, at concentrations low enough to avoid chromophore destruction, had no effect. Both large and small crude Avena sativa L. phytochrome showed two-component reversion kinetics.

EFFECTS OF ENVIRONMENTAL FACTORS ON MECHANICAL PROPERTIES OF THE CELL WALL IN RICE COLEOPTILES.

Cell wall properties determined by the stress-relaxation technique were studied with coleoptiles of rice seedlings grown under different environmental conditions. The cell wall was simulated by a viscoelastic model consisting of either four or an infinite number of Maxwell components. Reciprocal of relaxation time for the first component in the former model (1/τ1 ) and minimum and maximum relaxation times (To and Tm ) in the latter, in addition to the stress/strain ratio, were parameters representing cell wall properties. Parameters changed depending on the ages and regions of the coleoptilles used and 011 the environmental conditions under which rice seedlings were grown. Effects on cell wall properties of aeration during submerged growth, excision of the coleoptile tip, and exposure to small doses of red and/or far-red light were examined. In most cases, high values of 1/τ1 and of Tm and small values of To were consistent with the growth potentiality of cells, while the stress/strain ratio seemed to be a consequence of elongation growth.

Identification of auxin from Zea coleoptile tips by mass spectrometry

The auxin from Zea coleoptile tips has been identified conclusively as IAA.

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

AbstractEpiphytic, IAA‐producing bacteria strains were fed with 14C‐tryptophan (Try). 14C‐Try absorption and, after transfer to a Try‐free medium, 14C‐IAA output were stated. Using 4 different methods, the 14C‐Try containing bacteria were applied to the tips of sterile corn coleoptiles and the ‘diffusible’ auxin collected at the coleoptile bases by means of agar blocks. 14C‐IAA was detected in the agar blocks. Sterile coleoptiles the tips of which were wupplied with 14C‐Try also deliver some 14C‐IAA at their bases, but much less than both sterile coleoptiles supplied with 14C‐Try‐containing bacteria and nonsterile supplied with 14C‐Try.

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

AbstractMore “diffusible” auxin is received from nonsterile than from sterile corn coleoptile tips. An artificial reinfection of sterile coleoptiles with epiphytic, IAA‐producing bacteria strains does, a superinfection of nonsterile coleoptiles does not increase the auxin amount. The difference between sterile and nonsterile tips persists if diffusion from the coleoptile surface is excluded by covering the surface with a paraffin layer. The greater the distance from the apex, the higher becomes the superiority of nonsterile tips. An artificial bacterial contamination of the contact face between tip and receiver agar block, or addition of glucose and tryptophan to the agar block, do not influence the received auxin amount. Consequently the additional, bacteria‐produced auxin delivered by the nonsterile tip is not produced at the cut surface or in the agar but is present in the tissues of the coleoptile tip.

The Geoelectric Effect in Plant Shoots

The development of the geoelectric effect has been followed in Zea coleoptiles with a flowing solution electrode system, and its dependence upon auxin concentration gradients and aerobic metabolism assessed. A symmetrical source of IAA can effectively replace the coleoptile tip in allowing the geo electric potential to occur. The diffusate from coleoptile tips, when applied asymmetrically to the apex of a vertical decapitated coleoptile, generates a potential difference across the coleoptile indistinguishable from that induced by the asymmetrical application of IAA. Asymmetrical application of IAA to vertical Avena and Zea coleoptiles and Helianthus hypocotyls induces closely similar responses. Neither the geoelectric effect nor a geotropic response develops when intact Zea coleoptiles are placed horizontally after being deprived of oxygen, but they both occur when an aerobic atmosphere is restored. The lateral potential difference induced by the asymmetrical applica tion of IAA to the apex of a vertical coleoptile does not occur under anoxic conditions. With a static-drop electrode system and a decapitated Zea coleoptile, a potential difference develops immediately after reorientation of the coleoptile into the horizontal position, and attains a maximum value after about 10 min. This potential difference can be further increased by the asymmetrical application of IAA to the lower half of the apical cut surface of the coleoptile. Our data support the view that both the geoelectric potential and the geotropic response are due to the IAA concentration gradient which arises from the lateral transport of this substance from the upper to the lower half of the horizontal shoot. They also bear out our previous conclusions that the 'geoelectric potential' observed with static-drop electrodes and an intact shoot, is the resultant of two processes. The first is a physical phenomenon arising in the electrodes, or between the electrodes and the plant tissue, and the second arises in the living tissues of the shoot as the result of gravity-induced changes in auxin distribution.

Beiträge zur Physiologie der Morphaktinwirkung.: III. Hemmung des Auxintransports und Sulfhydrylaktivität

1. In pea seedlings, a ring of lanolin paste which contains the morphactin CFM reduces the IAA-dependent correlative inhibition of axillary buds, provided the ring is localized between the buds and the apical applied IAA. 2. CFM inhibits the basipetal IAA transport from donator agar blocks through sunflower hypocotyl sections into receiver agar blocks. 3. Gravity-induced curvatures of wheat coleoptile tips and IAA-induced curvatures of oat coleoptiles ( Avena standard curvature test) are altered by CFM, the length of the curved part of the coleoptile being greatly reduced. 4. In the Avena standard curvature test, CFM does not induce curvatures, but reduces IAA-induced curvatures. 5. CFM reduces the activity of the sulfhydryl enzyme urease. Cystein diminishes this reduction. 6. Cystein partially or completely counteracts the CFM effects on adventitious root initiation of Phaseolus hypocotyl cuttings. 7. It is concluded that CFM affects sulfhydryl-dependent reactions and consequently inhibits, among other things, active transport mechanisms and their sequential reactions.

Zea Shoot Development in Response to Red Light Interruption of the Dark-Growth Period. I. Inhibition of First Internode Elongation

Brief, low energy (approximately 400 Kerg cm(-2)) red light interruption of the early dark-growth period of Zea mays L. cv F-M Cross induced inhibition of first internode elongation which was maximal whenever light interruption occurred from 2.5 to 4 days of seedling age. Two-thirds of the maximal inhibition occurred in the tissues constituting the region 0 to 2 mm below the coleoptilar node at time of light treatment. The coleoptile tip showed the greatest sensitivity for red light reception in the internode response. Far-red light exposures following red light treatments reversed the red light effect. However, far-red light alone inhibited first internode elongation as effectively as red light of similar dosage.

Genetic Variation in Maize Coleoptile Growth and its Relationship to the Auxin Regulatory System1

Part of the variability of coleoptile elongation in a population of maize (Zea mays L.) was found to be due to genotypic differences. Genotypic differences were also found for five traits related to the auxin regulatory system. These five traits were (1) auxin production of corn coleoptile tips measured by Avena bioassay, (2) elongation of corn coleoptile sections without exogenous auxin, (3) elongation with exogenous auxin, (4) response to exogenous auxin measured by the differences between (3) and (2) above, and (5) peroxidase activity. Regression of coleoptile length after 5 days on the traits chosen to represent the auxin regulatory system resulted in a significant reduction in the sum of squares, and a coefficient of determination of 0.425.

INTERACTION OF INDOLYL ACETIC ACID AND GIBBERELLIC ACID IN THE SYNTHESIS OF α‐AMYLASE BY BARLEY ALEURONE

SummaryWhen slices of barley endosperm were supplied with a mixture of 10−4 M gibberellic acid and indolyl acetic acid (10−4 or 10−10 m) continuously for 36 hours the amount of α‐amylase formed was significantly less than that formed in response to gibberellic acid alone. However, pre‐treatment of slices with a mixture of 10−10 M indolyl acetic acid + gibberellic acid for 8 hours followed by incubation in gibberellic acid alone resulted in enhanced levels of production of α‐amylase.Removal of coleoptile tips from germinating grains was followed by diminished synthesis of α‐amylase in the endosperm, but the level of α‐amylase formed could be restored to that found in the intact grain by supplying 10−7 M indolyl acetic acid to the coleoptile stump.It is suggested that indolyl acetic acid, migrating from the coleoptile tip, is involved not only in the process of lignification in the scutellum but also in the preliminary stages of gibberellin‐mediated α‐amylase formation in the aleurone.

PHYTOCHROME ACTION IN ORYZA SATIVA L. III. THE SEPARATION OF PHOTOPERCEPTIVE SITE AND GROWING ZONE IN COLEOPTILES, AND AUXIN TRANSPORT AS EFFECTOR SYSTEM

1 In 4-day-old etiolated rice seedlings, 3 mm of the coleoptile tip did mainly perceive the photostimulus to cause the phytochrome-dependent inhibition of coleoptile elongation. At this age, cell elongation occurred most in the middle portion of coleoptiles in the dark, and was reversibly controlled by a brief exposure of the tip to red and far-red light. Thus, the photoperceptive site was evidently separated from the growing zone in intact rice coleoptiles. 2 The red-light-induced inhibition of coleoptile elongation was nullified by the removal of tip followed by the exogenous application of IAA. The sensitivity of thus treated coleoptiles to IAA was gradually lost during intervening darkness between the irradiation and the decapitation, and a 50% loss was obtained at ca. 6th hour at 26°C. 3 Polar auxin transport from coleoptile tips was remarkably prevented at the period between, at least, 2nd and 4th hour after red irradiation, and it recovered to the level of dark control by the 6th hour. Far-red light given immediately after red irradiation reversed the yield of diffusible auxin up to that of far-red control.

GEOTROPIC CURVATURE OF AVENA COLEOPTILES IN SOLUTIONS OF VARIOUS OSMOTIC VALUES

Isolated Avena coleoptiles are placed with their bases in mannitol solutions of various osmotic values. In this way the water uptake is inhibited. The coleoptiles are irradiated with red light and turned through 90 °. Considerable geotropic curvatures are developed in solutions which prevent growth to a large extent. Red light enhances the geotropic response also under these conditions, but a red, far-red reversal of the red effect does not take place. The tips of the horizontal coleoptiles appear not to reach the vertical position, but end up growing at a constant angle to the vertical.

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

AbstractGibberellic 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.

Gravitational Compensation and the Phototropic Response of Oat Coleoptiles

Avena seedlings were germinated and grown while continuously rotated on the horizontal axis of a clinostat. The coleoptiles of these gravity-compensated plants were phototropically more responsive than those of plants rotated on a vertical axis. When the plants were compensated after unilateral irradiation, phototropic curvature of the shoot progressed for the next 6 hours, with the rate of curving decreasing about 3 hours after irradiation. The decrease in rate was less in the plants gravity-compensated before irradiation than in those vertically rotated. In the period 70 to 76 hours after planting, the growth rate of the compensated coleoptiles was significantly less than that of the vertically rotated seedlings. The greater phototropic curvature, the decreased growth rate, and the slower rate of straightening of the curved, compensated shoot can be correlated with several consequences of compensation: an increase in sensitivity to auxin, a lowering of auxin content in the coleoptile tip, and possibly, from an interaction between compensation and phototropic stimulation, an enhanced difference in auxin transport between the illuminated and shaded halves of the unilaterally irradiated shoot.The phototropic response of the vertically rotated seedling was significantly different from that of the vertical stationary, indicating the importance of vertically rotated controls in clinostat experiments.

Auxin Transport in Zea mays Coleoptiles II. Influence of Light on the Transport of Indoleacetic Acid-2-14C

The effect of bilateral irradiation with white light (1000 Meter Candle Sec) on the basipetal transport of auxin has been investigated. Illumination of either the intact shoot or the excised coleoptile tip of the Zea seedling, decreased the amount of diffusible auxin obtained from the tip, and decreased Avena curvature response to unilaterally applied indoleacetic acid. Irradiation of the intact Zea seedling did not affect the absorption of (14)C-labeled indoleacetic acid from an agar block subsequently placed on the decapitated coleoptile. However, light caused a significant decrease in the amount of labeled auxin basipetally transported, without affecting materially the velocity of that transport. These and other observations are interpreted as support for the hypothesis that the primary hormonal phenomenon in first-positive phototropism is a light-induced impairment in the basipetal transport of auxin.

The Physiological Versus the Spectrophotometric Status of Phytochrome in Corn Coleoptiles

The influence of red light in altering the phototropic sensitivity of corn coleoptiles (Zea mays L., cultivar Burpee Barbecue Hybrid) is compared with the spectrophotometric status of the phytochrome they contain. The distribution of measurable phytochrome corresponds roughly with the distribution of sensitivity to red light for physiological change. Both phytochrome concentration and red light sensitivity are maximal in the coleoptile tips. Red light pretreatments which reduce total phytochrome by about 50%, however, do not alter subsequent red light sensitivity of the phototropic system. Dosages of red light sufficient to saturate the physiological system are two orders of magnitude too small to induce measurable phytochrome transformation. The log-dosage-response curves for physiological change and for phytochrome transformation do not have the same slopes. The time course for appearance, mainconcentration of the far-red-absorbing form of phytochrome over a broad range of tenance, and decay of the physiological response is independent of the measurable concentrations. The following hypothesis is proposed: the phytochrome mediating the alteration in phototropic sensitivity is only a small proportion of the total present. This small active fraction is physically and kinetically independent of the bulk measurable, and is packaged in some manner which facilitates its transformation in both directions.

Hormonal Relations in the Phototropic Response IV. Light-Induced Changes of Endogenous Auxins in the Coleoptile

Beside indoleacetic acid (IAA), 3 auxins were found by chromatographic resolution of acidic fractions of Avena and Zea coleoptile tips. One of these auxins, designated P, occurred at levels of activity approaching those of IAA. The other 2 auxins, termed F and M, occurred at lower levels of activity. When the auxins of the excised coleoptile tips were isolated immediately after equilateral or unilateral irradiation with blue light at first positive energies, the ratio of IAA to the other auxins increases. This rise is the result of a decrease in P and F, and probably an increase in IAA. Light did not affect materially the total auxin content. It is suggested that P and F might be associated with the basipetal transport inequalities of IAA in phototropism.P has been partially characterized. Its R(F) on chromatograms developed in ammoniacal isopropanol is about 0.65. It is converted to IAA in vitro by heat. The ultraviolet absorption spectrum of chromatographically resolved P also suggests an indolyl complex. P is not readily transported basipetally, and the slope of its relative concentration-response curve (Avena section test) is lower than that of IAA. P does not appear to be any of the chemically characterized native auxins.

Role of Indole-3-acetic Acid in Modification of Geotropic Responses in Clinostat Rotated Avena Seedlings

Oat seedlings were grown in a sand medium on clinostats with horizontal axes of rotation to nullify the directional component of the gravity-force vector. Coleoptile segments from such seedlings showed an enhanced absorption of apically applied exogenous auxin (indole-3-acetic acid), compared to segments from vertically rotated or stationary controls. Absorption of basally applied auxin and auxin transport were unaffected by the gravity treatments. Horizontal rotation did not materially change the amount of auxin produced and transported from excised coleoptile tips: however, plants so rotated showed an enhanced curvature response to unilaterally applied auxin.Collectively, these experiments indicate that enhanced plant responses to horizontal clinostat rotation, where rates of rotation are sufficient to nullify the directional component of the gravity-force vector, are caused primarily by increases in metabolism and not by a modification of auxin availability. These data do not support recently advanced hypotheses that the polarity of auxin transport is based on gravitational sedimentation of cell inclusions.

The Effects of Paraffin Oil on Phototropic and Geotropic Responses in Avena Coleoptiles

Experiments are described which were designed to test the significance of the coleoptile tip as the site of reception of light stimulus leading to negative photo-tropic response under paraffin oil. The results show clearly that the tip is of paramount importance in this respect. Further experiments in which the coleoptile tips were bisected in a plane at right angles to the light rays lend support to the hypothesis that negative phototropism under oil is related tolateral transport of material in the tip. Lastly, experiments which show that the geotropic response of coleoptiles is not reversed by immersion in oil are described. These findings are discussed in relation to certain hypotheses concerning the mechanism of negative phototropism under oil.

Hormonal Relations in the Phototropic Response: III. The Movement of C14-labeled and Endogenous Indoleacetic Acid in Phototropically Stimulated Zea Coleoptiles

The effect of phototropic stimulation of Zea coleoptile tips on the distribution of both endogenous indoleacetic acid (IAA) and applied C(14)-labeled IAA was determined. The tips rested on bisected agar blocks. More IAA was found in the blocks under the shaded side of the coleoptile tips than those under the irradiated side. However, no significant difference was observed between the total amounts of IAA, endogenous or labeled, in the irradiated and shaded sides of the experimental system. In addition, less endogenous auxin was found in the shaded tissues than in their irradiated counterparts. It is suggested that phototropism following unilateral irradiation with first positive radiant densities might be a consequence of lateral inequalities in the ability of the irradiated and shaded tissues to transport auxin basipetally.