Effects of Ethylene on the Kinetics of Curvature and Auxin Redistribution in Gravistimulated Roots of Zea mays

We tested the involvement of ethylene in maize (Zea mays L.) root gravitropism by measuring the kinetics of curvature and lateral auxin movement in roots treated with ethylene, inhibitors of ethylene synthesis, or inhibitors of ethylene action. In the presence of ethylene the latent period of gravitropic curvature appeared to be increased somewhat. However, ethylene-treated roots continued to curve after control roots had reached their final angle of curvature. Consequently, maximum curvature in the presence of ethylene was much greater in ethylene-treated roots than in controls. Inhibitors of ethylene biosynthesis or action had effects on the kinetics of curvature opposite to that of ethylene, i.e. the latent period appeared to be shortened somewhat while total curvature was reduced relative to that of controls. Label from applied ³H-indole-3-acetic acid was preferentially transported toward the lower side of stimulated roots. In parallel with effects on curvature, ethylene treatment delayed the development of gravity-induced asymmetric auxin movement across the root but extended its duration once initiated. The auxin transport inhibitor, 1-N-naphthylphthalamic acid reduced both gravitropic curvature and the effect of ethylene on curvature. Since neither ethylene nor inhibitors of ethylene biosynthesis or action prevented curvature, we conclude that ethylene does not mediate the primary differential growth response causing curvature. Because ethylene affects curvature and auxin transport in parallel, we suggest that ethylene modifies curvature by affecting gravity-induced lateral transport of auxin, perhaps by interfering with adaptation of the auxin transport system to the gravistimulus.     

The “acid growth effect” and geotropism

Summary The curvature developed by segments of sunflower hypocotyl exposed to gravitational stimulus was enhanced in buffer solutions between pH 3.4 and 4.0 in the absence of added auxin. This effect was observed both when the segments were submerged during the stimulus and when they floated near the surface of the solution. 5–10 min in a horizontal position was sufficient to induce subsequent curvature.Straight growth of the segments was also promoted in buffers of this pH range.The acid effect on curvature was insensitive to KAsO₂, HgCl₂ and cycloheximide, inhibitors which drastically reduced auxin-induced curvature. Furthermore, acid buffer, but not auxin, restored the ability of segments taken from etiolated and starved plants to respond to gravity. These results suggest that the polarisation following gravistimulus may not be resticted to the asymmetric distribution of auxin and auxin co-factors but may involve a general physiological asymmetry.     

Effects of indole-3-acetic acid and auxin transport inhibitors on the style curvature of three Alpinia species (Zingiberaceae)

The effects of indole-3-acetic acid (IAA) and the auxin transport inhibitors 2, 3, 5-triiodobenzoic acid (TIBA) and 1-N-naphthylphthalamic acid (NPA) on the style curvature of Alpinia platychilus, A. blepharocalyx, and A. mutica were studied. Exogenous IAA stimulated the style curvature movement of the anaflexistylous morph (ana-morph) and cataflexistylous morph (cata-morph) of three Alpinia species in light, but had no effect in the dark. Treatment with auxin efflux inhibitors (NPA and TIBA) before flower opening did not affect the first curvature of the two morphs in darkness; however, the subsequent second movement of the ana-morph was enhanced by NPA or TIBA, while the second movement of the cata-morph was completely inhibited. After the first curvature, NPA and TIBA treatments at 06:00?hours (before significant illumination) and 11:00?hours (after the styles were illuminated for 4?h) increased the second curvature of the ana-morph, but significantly decreased that of the cata-morph. The effect at 06:00?hours was more significant than the effect at 11:00?hours. These results suggested that auxin and auxin transport affected the style curvature in a different way in the two morphs, and two morphs had distinct mechanisms for style movement at different times.     

Ethylene Interacts with Auxin in Regulating Developmental Attenuation of Gravitropism in Flax Root

Previous research shows that gravity-sensing in flax (Linum usitatissimum) root is initiated during seed imbibition and precedes root emergence. In this study we investigated the developmental attenuation of flax root gravitropism post-germination and the involvement of ethylene. Gravity response deteriorated significantly from 3 to 11?h after root emergence, which occurred at around 19?h after imbibition (that is, from “age” 22 to 30?h). Although the root elongation rate increased from 22 to 30?h, the gravitropic curving rate declined steadily. Older roots were able to tolerate higher levels of exogenous IAA before inhibition of elongation and gravitropism occurred. The age-dependent effect of IAA on root growth and gravitropism suggests that young roots are more sensitive to auxin and respond to a smaller vertical auxin gradient than older roots upon horizontal gravistimulation. The ethylene synthesis inhibitor AVG (2-aminoethoxyvinyl glycine, 10?μM) or ethylene action inhibitor Ag⁺ (10?μM) stimulated gravitropic curvature of 30?h roots by 24 and 32%, respectively, but had no effect on 22?h roots, suggesting that as roots age, ethylene begins to play a role in root gravitropism. The auxin transport inhibitor NPA (N-naphthylphthalamic acid, 50?μM) reduced gravitropic curvature of 30?h roots by 24% but had no effect on 22?h roots. On the other hand, treating roots simultaneously with the auxin transport inhibitor and ethylene synthesis or action inhibitor stimulated gravitropic curvature of 30?h roots but not 22?h roots. Taken together, these data indicate that as roots develop, their weakened gravity response is due to decreased auxin sensitivity and possibly auxin transport regulated by ethylene.     

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

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 ¹⁴C-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 EFFECT OF AUXINS UPON PROTOPLASMIC STREAMING

1. Evidence has accumulated that the action of auxins in promoting growth is exerted not upon the cell wall but upon the cell contents; i.e., the protoplasm. Following indications previously obtained, therefore, the effect of auxins on the rate of protoplasm streaming in the Avena coleoptile was studied. 2. Indole-3-acetic acid, the most active auxin available in pure form, was found to increase the rate of streaming in the epidermal cells of the Avena coleoptile at concentrations between 0.5 and 0.002 mg. per liter, the maximum increase being brought about at 0.01 mg. per liter. This concentration is approximately that which, applied in agar to one side of the decapitated coleoptile, would give a curvature of 1°; i.e., it is well within the range of concentrations active in growth promotion. It is, however, much less than that which produces maximum elongation in immersed sections of Avena coleoptiles. 3. This accelerating effect is readily determined quantitatively by comparison with the streaming in control coleoptiles in pure water, which, if thoroughly aerated, maintain a constant rate for over an hour. The accelerating effect takes place immediately and is over within about 30 minutes. 4. Concentrations of indole-3-acetic acid greater than 0.5 mg.per liter inhibit the streaming, the effect being also over in about 30 minutes, and its extent increasing with increasing auxin concentration. This parallels the effect of high auxin concentrations in inhibiting elongation, although the inhibition of streaming is obtained at much lower concentrations than inhibit elongation. 5. The effects of indole-3-acetic acid on streaming are not specific for that substance, but appear to be common to auxins in general. Thus coumaryl-3-acetic acid and allocinnamic acid, both of which bring about cell enlargement, root formation, and bud inhibition, i.e. are typical auxins, also cause an immediate acceleration of the rate of streaming, and as with indole-acetic add the effect is over in about 30 minutes. The concentrations of these two substances which produce the maximum effect are about ten times that of indole-acetic acid, which approximately corresponds with their relative auxin activities. The curves relating concentrations of these substances to their effects on streaming are very similar to that for indole-acetic acid. 6. On the other hand, certain substances which are known to affect streaming in other materials do not produce any effect comparable to that of auxin. Ethylene chlorhydrin, histidine, and urea in all concentrations were without effect on streaming in the Avena coleoptile within the first 30 minutes of treatment. 7. The effects produced by the auxins were not due to pH. 8. The action on streaming here studied is evidently quite different from the re-starting of streaming after its cessation, studied by Fitting in Vallisneria. Correspondingly histidine, which in Fitting''s experiments showed activity down to 10^–7 M, is inactive here. 9. Per contra, the effect of auxin here studied is on normal streaming. It takes place immediately and at concentrations in the same range as those which produce growth. The curve of effect against concentration parallels that for growth although the actual concentration values differ. It is therefore reasonable to suppose that the effect of auxin on streaming is closely connected with one of the first stages of its effect on the growth process.     

Root Nodule Symbiosis II

Nodule-roots of Myrica cerifera (Southern Wax Myrtle) and Casuarina cunning hamiana (Australian Pine) have a negative geotropic curvature. Studies of their endogenotts auxin content revealed a pattern of correlation: the absence of detectable auxin when the geotropisni was negative. Non-nodulated roots of Myrica exhibited a normal positive geotropic curvature and possessed an auxin content within an anticipated range (10 mg/kg). Root nodules of Alnus species, whose roots exhibit a positive geotropic curvature, contained measurable endogenous auxin (20 mg IAA/kg). The presence of an indoleaectic acid oxidase system in Myrica and Casuarina root nodules has heen described and correlations are drawn between non-detectable endogenous auxin concentrations and high enzymatic activities. It is suggested that the negative geotropic curvature of the nodule-roots of Myrica and Casuarina is due to the presence of a sub-optimal concentration of auxin which in turn results from the activity of an indoleacetic aeid destroying system.     

Effects of Cations on Hormone Transport in Primary Roots of Zea mays

We examined the influence of aluminum and calcium (and certain other cations) on hormone transport in corn roots. When aluminum was applied unilaterally to the caps of 15 mm apical root sections the roots curved strongly away from the aluminum. When aluminum was applied unilaterally to the cap and ³H-indole-3-acetic acid was applied to the basal cut surface twice as much radioactivity (assumed to be IAA) accumulated on the concave side of the curved root as on the convex side. Auxin transport in the apical region of intact roots was preferentially basipetal, with a polarity (basipetal transport divided by acropetal transport) of 6.3. In decapped 5 mm apical root segments, auxin transport was acropetally polar (polarity = 0.63). Application of aluminum to the root cap strongly promoted acropetal transport of auxin reducing polarity from 6.3 to 2.1. Application of calcium to the root cap enhanced basipetal movement of auxin, increasing polarity from 6.3 to 7.6. Application of the calcium chelator, ethylene-glycol-bis-(β-aminoethylether)-N,N,N′, N′-tetraacetic acid, greatly decreased basipetal auxin movement, reducing polarity from 6.3 to 3.7. Transport of label after application of tritiated abscisic acid showed no polarity and was not affected by calcium or aluminum. The results indicate that the root cap is particularly important in maintaining basipetal polarity of auxin transport in primary roots of corn. The induction of root curvature by unilateral application of aluminum or calcium to root caps is likely to result from localized effects of these ions on auxin transport. The findings are discussed relative to the possible role of calcium redistribution in the gravitropic curvature of roots and the possibility of calmodulin involvement in the action of calcium and aluminum on auxin transport.     

Can Lateral Redistribution of Auxin Account for Phototropism of Maize Coleoptiles?

Elongation growth of intact, red-light grown maize (Zea mays L.) coleoptiles was studied by applying a small spot of an indole acetic acid (IAA)-lanolin mixture to the coleoptile tip. We report that: (a) endogenous auxin is limiting for growth, (b) an approximately linear relation holds between auxin concentration and growth rate over a range which spans those rates occurring in phototropism, and (c) an auxin gradient established at the coleoptile tip is well sustained during its basipetal transport. We argue that the growth differential underlying coleoptile phototropism (first-positive curvature) can be explained by redistribution of auxin at the coleoptile tip.     

THE INTERACTION OF AUXIN AND ETHYLENE IN THE MAINTENANCE OF LEAF BLADE FORM IN PHASEOLUS VULGARIS L. VAR. PINTO

Auxin treatment results in hyponastic curvature of the primary leaves of Phaseolus vulgaris L. var pinto. Ethylene production by hyponastic leaves is detected within 1 hr after treatment with IAA in concentrations at or above 1 μm. The amount of ethylene detected is proportional to the concentration of auxin applied. Untreated control leaves and leaves treated with 2,3,5-tri-iodobenzoic acid or gibberellic acid did not produce ethylene detectable by our equipment. The hyponastic curvature induced by auxin treatment can be inhibited by exogenous application of ethylene or ethylene-generating compounds, and these treatments produce epinasty in auxin-treated leaves. Treatment with inhibitors of ethylene synthesis or action, such as aminoethoxy-vinylglycine, carbon dioxide, or heat treatment, prolong hyponasty. The planar form, therefore, appears to be affected by both hyponastic auxin effect and an epinastic ethylene effect.     

The Role of Starch in the Gravitropic Response of the Lentil Root

It is well accepted that the amyloplasts of the cap are responsible for gravisensing in primary roots. However, roots with starch-depleted plastids are able to respond to gravistimulus, but their curvature is slower than that of roots containing amyloplasts. The goal of our experiment was to analyse the effects of natural variations of statolith starch in the gravitropic response of lentil roots to a stimulation in the horizontal position. In lentil seedlings grown in the vertical position for 26 h, the volume of the amyloplasts in the statocytes differed between individual roots. The amount of starch in the cap was determined parallel to the rate of gravitropic curvature. There was no statistical correlation between the intensity of the gravitropic response and the starch content in the statocytes. Lentil roots were treated with gibberellic acid (GA₃) at 32°C in order to reduce the volume of starch in the statoliths. There was 53% less starch in the cap of GA₃treated roots as compared to the cap of control roots. But there was no relationship between starch content in the cap and the responsiveness of the root to a gravistimulus, except when the amount of starch was small.     

THE TIMING OF AND EFFECT OF TEMPERATURE ON AUXIN-INDUCED HYPONASTIC CURVATURE OF THE BEAN PRIMARY LEAF BLADE

Detailed examination of the hyponastic curvature of the primary bean leaf blade in response to indoleacetic acid (IAA) shows that curvature begins within 15 min after application and increases to a maximal rate at 20 to 30 min. A second application of IAA results in a second curvature maximum when applied 1.5 hr or more after the first. Washing experiments indicate IAA uptake is largely complete by about 20 min after application, suggesting the return to planar form is accompanied by the uptake and passage of a wave of IAA through the responding cells. The rate of curvature decreases as the temperature is lowered, particularly below 14 C; at low concentrations (10^–4 m) the rate of response to 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxypropionic acid is slower than that for IAA and naphthaleneacetic acid. These differences are proposed to reflect the involvement of the polar auxin transport system in the response. The leaves of bean seedlings exposed to 4 C develop hyponastic curvatures when returned to normal growth temperature; 5 min treatment is sufficient to induce this response, and with longer treatments, greater curvatures are obtained. This curvature is inhibited by application of 2,3,5-triiodobenzoic acid (TIBA) to the undersurface of the leaf at the beginning of the cold treatment. The results are consistent with a model of planar plageotropic growth regulation in the leaf blade in which auxin produced by cells in the upper portion of the blade is transported by the polar transport system through cells in the lower portion that are growth limited by auxin supply. The hyponastic and epinastic effects caused by exogenous application of auxin or TIBA and of cold treatments are considered to result from changes in this auxin supply.     

Evidence for a Relationship between H Excretion and Auxin in Shoot Gravitropism

The role of auxin and protons in the gravitropic response of the sunflower (Helianthus annuus L. cv Sungold) hypocotyl has been investigated. No physiological asymmetry in acid-growth capacity could be detected between the upper and lower surfaces of gravistimulated hypocotyls. These data imply that neutral buffers inhibit shoot gravitropism by preventing the establishment of a lateral proton gradient along gravitropically stimulated hypocotyls. Indirect evidence that auxin is involved in the establishment and/or maintenance of such a gradient derives from the quantitative assessment of the effects of exogenous auxin, anti-auxins, and vanadate on gravicurvature. At low concentrations, exogenous auxin accelerated curvature; at high concentrations, curvature was prevented. Vanadate, an inhibitor of auxin-enhanced H⁺ secretion, α-(p-chlorophenoxy)isobutyric acid (PCIB), an anti-auxin, and 2,3,5-triiodobenzoic acid (TIBA), an auxin-transport inhibitor, prevented observable asymmetric proton excretion using a brom cresol purple agar technique and also inhibited gravicurvature. Vanadate, PCIB, and TIBA inhibition of gravicurvature could be reversed with acid treatment to the lower surface of a gravistimulated hypocotyl. Auxin treatment to the lower surface of a gravistimulated hypocotyl did not reverse vanadate-induced inhibition, but it did partially reverse PCIB- and TIBA-induced inhibition. These results indicate a close relationship between the acid-growth theory and the differential growth responses of the sunflower hypocotyl during gravitropism.     

Correlations between Gravitropic Curvature and Auxin Movement across Gravistimulated Roots of Zea mays

We compared the kinetics of auxin redistribution across the caps of primary roots of 2-day-old maize (Zea mays, cv Merit) seedlings with the time course of gravitropic curvature. [³H] indoleacetic acid was applied to one side of the cap in an agar donor and radioactivity moving across the cap was collected in an agar receiver applied to the opposite side. Upon gravistimulation the roots first curved upward slightly, then returned to the horizontal and began curving downward, reaching a final angle of about 67°. Movement of label across the caps of gravistimulated roots was asymmetric with preferential downward movement (ratio downward/upward = ca. 1.6, radioactivity collected during the 90 min following beginning of gravistimulation). There was a close correlation between the development of asymmetric auxin movement across the root cap and the rate of curvature, with both values increasing to a maximum and then declining as the roots approached the final angle of curvature. In roots preadapted to gravity (alternate brief stimulation on opposite flanks over a period of 1 hour) the initial phase of upward curvature was eliminated and downward bending began earlier than for controls. The correlation between asymmetric auxin movement and the kinetics of curvature also held in comparisons between control and preadapted roots. Both downward auxin transport asymmetry and downward curvature occurred earlier in preadapted roots than in controls. These findings are consistent with suggestions that the root cap is not only the site of perception but also the location of the initial redistribution of effectors that ultimately leads to curvature.     

Physiology of Movements in the Stems of Seedling Pisum sativum L. cv Alaska : III. Phototropism in Relation to Gravitropism, Nutation, and Growth

Phototropic response in etiolated pea (Pisum sativum L. cv Alaska) seedlings is poor. However, the curvature induced by unilateral blue light can be hastened and increased in magnitude by a previously administered red light pulse followed by several hours of darkness. Phytochrome is involved in the red light effect. Phototropic response was almost completely inhibited by removal of the apical bud and hook, but it was restored if exogenous indole-3-acetic acid was applied apically to the cut stump. Therefore, the stem contains both the phototropic photoreceptor and response mechanism. Perception of gravity and gravitropic response were also localized in the stem, but gravitropism was scarcely inhibited by decapitation. It was also observed that the kinetics and curvature pattern of gravitropism differed greatly from those of phototropism. Like phototropism, stem nutation required auxin and was promoted by red light. Unlike phototropism, photoenhanced nutational curvature required the apical hook and was propagated as a wave down the stem. Naphthylphthalamic acid inhibited, in order of decreasing effect, nutation, phototropism/gravitropism, and growth. Phototropism, gravitropism, and nutation appear to represent distinct forms of stem movement with fundamental differences in the mechanisms of curvature development.     

Electrotropism of Maize Roots : Role of the Root Cap and Relationship to Gravitropism

We examined the kinetics of electrotropic curvature in solutions of low electrolyte concentration using primary roots of maize (Zea mays L., variety Merit). When submerged in oxygenated solution across which an electric field was applied, the roots curved rapidly and strongly toward the positive electrode (anode). The strength of the electrotropic response increased and the latent period decreased with increasing field strength. At a field strength of 7.5 volts per centimeter the latent period was 6.6 minutes and curvature reached 60 degrees in about 1 hour. For electric fields greater than 10 volts per centimeter the latent period was less than 1 minute. There was no response to electric fields less than 2.8 volts per centimeter. Both electrotropism and growth were inhibited when indoleacetic acid (10 micromolar) was included in the medium. The auxin transport inhibitor pyrenoylbenzoic acid strongly inhibited electrotropism without inhibiting growth. Electrotropism was enhanced by treatments that interfere with gravitropism, e.g. decapping the roots or pretreating them with ethyleneglycol-bis-[β-ethylether]-N,N,N′,N′-tetraacetic acid. Similarly, roots of agravitropic pea (Pisum sativum, variety Ageotropum) seedlings were more responsive to electrotropic stimulation than roots of normal (variety Alaska) seedlings. The data indicate that the early steps of gravitropism and electrotropism occur by independent mechanisms. However, the motor mechanisms of the two responses may have features in common since auxin and auxin transport inhibitors reduced both gravitropism and electrotropism.     

Transport of Indole-3-Acetic Acid during Gravitropism in Intact Maize Coleoptiles

We have investigated the transport of tritiated indole-3-acetic acid (IAA) in intact, red light-grown maize (Zea mays) coleoptiles during gravitropic induction and the subsequent development of curvature. This auxin is transported down the length of gravistimulated coleoptiles at a rate comparable to that in normal, upright plants. Transport is initially symmetrical across the coleoptile, but between 30 and 40 minutes after plants are turned horizontal a lateral redistribution of the IAA already present in the transport stream occurs. By 60 minutes after the beginning of the gravitropic stimulus, the ratio of tritiated tracer auxin in the lower half with respect to the upper half is approximately 2:1. The redistribution of growth that causes gravitropic curvature follows the IAA redistribution by 5 or 10 minutes at the minimum in most regions of the coleoptile. Immobilization of tracer auxin from the transport stream during gravitropism was not detectable in the most apical 10 millimeters. Previous reports have shown that in intact, red light-grown maize coleoptiles, endogenous auxin is limiting for growth, the tissue is linearly responsive to linearly increasing concentrations of small amounts of added auxin, and the lag time for the stimulation of straight growth by added IAA is approximately 8 or 9 minutes (TI Baskin, M Iino, PB Green, WR Briggs [1985] Plant Cell Environ 8: 595-603; TI Baskin, WR Briggs, M Iino [1986] Plant Physiol 81: 306-309). We conclude that redistribution of IAA in the transport stream occurs in maize coleoptiles during gravitropism, and is sufficient in degree and timing to be the immediate cause of gravitropic curvature.     

Unilateral reorientation of microtubules at the outer epidermal wall during photo- and gravitropic curvature of maize coleoptiles and sunflower hypocotyls

Auxin (indole-3-acetic acid) controls the orientation of cortical microtubes (MT) at the outer wall of the outer epidermis of growing maize coleoptiles (Bergfeld, R., Speth, V., Schopfer, P., 1988, Bot. Acta 101, 57-67). A detailed time course of MT reorientation, determined by labeling MT with fluorescent antibodies, revealed that the auxin-mediated movement of MT from the longitudinal to the transverse direction starts after less than 15 min and is completed after 60 min. This response was used for a critical test of the functional involvement of auxin in tropic curvature. It was found that phototropic (first phototropic curvature) as well as gravitropic bending are correlated with a change of MT orientation from transverse to longitudinal at the slower-growing organ flank whereas the transverse MT orientation is maintained (or even augmented) at the faster-growing organ flank. These directional changes are confined to the MT subjacent to the outer epidermal wall. The same basic results were obtained with sunflower hypocotyls subjected to phototropic or gravitropic stimulation. It is concluded that auxin is, in fact, involved in asymmetric growth leading to tropic curvature. However, our results do not allow us to discriminate between an uneven distribution of endogenous auxin or an even distribution of auxin, the activity of which is modulated by an unevenly distributed inhibitor of auxin action.     

GROWTH AND GRAVITATIONAL RESPONSE IN THE DEVELOPMENT OF LEAF BLADE HYPONASTY

Rapid (4 hr) auxin-induced hyponastic curvature of primary leaves of Phaseolus vulgaris is shown to depend on a positive increase in growth of the lower portion of the blade. The curvature involves laminar growth as well as vein growth and is not due to simple turgor changes. The response is sensitive to gravitational orientation, as inversion and horizontal rotation reduce the auxin-induced curvature. The ethylene-generating compound, 2-chloroethylphosphonic acid, had no hyponastic effect on the leaves when applied to either the upper or lower surface and it inhibited auxin-induced hyponasty. This inhibition was additive to that of inversion. Long-term (24–48 hr) effects of 1 mM auxin depend on the surface of the leaf treated. Application to the upper surface results in epinasty, lower surface application in hyponasty, although the initial response in each case is a hyponastic curvature. A dorsi-ventral auxin transport system and differential auxin sensitivity of upper and lower portions of the leaf blade are postulated to account for these responses.     

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.