Ethylene modification of an auxin pulse in cotton stem sections

The effect of ethylene on the basipetal movement of indole-3-acetic acid-1-¹⁴C through cotton stem sections (Gossypium hirsutum, L. var. Stoneville 213) was studied apart from processes involved in the uptake and exit of auxin by the section. Stem sections 60 mm in length were pretreated with ethylene or placed in room air (control) and pulse labeled for 20 min with IAA-1-¹⁴C. In both the ethylene treated and control sections, the IAA-1-¹⁴C taken up moved basipetally as a peak of radioactivity. Generally, the applied pulse moved down the stem sections at an average velocity of approximately 5.8 mm per hr. In some experiments, however, ethylene slightly reduced the velocity of auxin transport. Although the peak of radioactivity became broader and more dispersed during its migration through the section, it was still distinguishable after 7 hr of transport.     

Movement of Pulses of Labeled Auxin in Corn Coleoptiles

The transit of indole-3-acetic acid through 20-mm sections of corn coleoptiles can be separated from processes involved in the uptake of auxin by the section and the exit of auxin from the section. Aerobic sections are supplied with an exogenous source of (14)C IAA for a limited time, and after the source is removed, a pulse of (14)C IAA moves down at 12 to 15 mm/hour. After transfer to nitrogen, movement of the pulse at the aerobic rate persists for about 10 minutes; thereafter drops to only 1 to 2 mm/hour and remains at this level during the next 4 hours. Within 2 hours, 70% of the total (14)C in aerobic sections has moved 10 mm or more down the section from the position of the initial peak, whereas after the same time in nitrogen less than 10% of the total (14)C has moved as far.During the migration down the coleoptile, the peak of radioactivity becomes broader and less distinct. This dispersion is more rapid in aerobic than anaerobic sections, but appears to be nonpolar and to occur along the existing concentration gradients. Diffusion probably contributes to this dispersion.In both inhibited and uninhibited sections, the movement of the peak, in contrast to its dispersion, is A) polar (downward) and B) independent of existing concentration gradients. Thus transit within the section possesses the fundamental properties of the overall transport system. The reduced amount of transport in inhibited sections is more likely maintained by glycolysis than by a low level of aerobic respiration dependent on the residual oxygen in the tissue.     

Time course of auxin stimulations of growth

Measurements of the time course of growth responses of corn coleoptile sections to pulses of auxin (10⁻⁵m indoleacetic acid) establish that the growth rate changes in a regular pattern around the auxin pulse: a latent phase of 12 to 15 minutes is followed by an acceleration of growth rate lasting 15 to 20 minutes, after which a fairly steady rate is maintained. When the auxin source is withdrawn, there is an after-effect of about 15 minutes followed by a decay of growth rate, which reaches 50% decay after a further 15 to 40 minutes. The decay phase appears to be a function of the transport of auxin out of the sections. The 50% decay of growth for single cells is estimated at 30 minutes from the time of withdrawal of an exogenous supply of auxin. The regulation of growth by auxin is rapidly imposed or dissipated as auxin enters and exits, respectively, suggesting a facile association and disassociation of auxin with a growth-limiting site in the cell. It is proposed that the growth-stimulated state is dissipated at once when the transportable auxin has passed out of the cell.     

Regulation of Auxin Levels in Coleus blumei by Ethylene

An investigation of the effects of ethylene pretreatment on several facets of auxin metabolism in Coleus blumei Benth “Scarlet Rainbow” revealed a number of changes presumably induced by the gas. Transport of indoleacetic acid-1-¹⁴C in excised segments of the uppermost internode was inhibited by about 50%. Decarboxylation of indoleacetic acid-1-¹⁴C by enzyme breis was not affected by the pretreatment. Levels of extractable native auxin in upper leaf and apical bud tissue of the pretreated plants were approximately one-half of those present in untreated plants. The rate of formation of auxin from tryptophan by enzyme breis from pretreated plants was approximately one-half that occurring in incubation mixtures containing the enzyme system from untreated plants. The conjugation of indoleacetic acid-1-¹⁴C in a form characterized chromatographically as indoleacetylaspartic acid was increased 2-fold in the upper stem region of plants pretreated with ethylene.     

On the Question of Stem Polarity with Respect to Auxin Transport

The effect of section length and number of longitudinally contiguous cells upon polar transport of natural auxin from the pine stem cambial region was investigated with oat coleoptile curvature tests. Basipetal and acropetal efflux of auxin to agar declines with increasing length of the sections, but the polarity quotient varies little and is similar to the polarity of individual cells. An integrated system of cells produces a wave along the stem in the efflux of auxin from consecutive segments. The possible role of such waves in development of polarity gradients and of the morphogenic maps of orientation of cells in the stem cambial region is discussed.     

Transient effects of light on auxin transport in the Avena coleoptile

The transport of indole-3-acetic acid-1-¹⁴C (IAA) through 4 mm segments of etiolated Avena coleoptiles was studied as a function of time by applying IAA in apical agar blocks and measuring the basal IAA export rate at 5-minute intervals. The transport velocity found in this way is at least 15 mm/hour at 26°. Following a 30-minute equilibration period, the export rate is nearly constant for at least 50 minutes at physiological donor concentrations. Exposure to about 3 × 10⁵ ergs/cm² blue light for 15 minutes leads to a transient reduction in the export. The export rate reaches a minimum about 25 minutes after the onset of illumination, then rises to reach a maximum by 35 minutes, and subsequently declines again. The result is a net export depression during the first 80 minutes, amounting to some 12 to 17%. Its timing closely matches the timing of the light growth response elicited by the same light dosage.

At higher IAA concentrations (0.5 and 1.8 mg/l), the export rate reaches a peak about 60 minutes after the initial application of auxin, and thereafter declines rapidly. Light increases this decline in export rate, without causing peaks and troughs, and even at 0.25 mg/l IAA the transient changes in export appear to be superimposed on a gradual decline in export rate after illumination. Blue light is effective in all these phenomena; the red far-red system appears to exert no effect. The results are discussed in relation to the mechanism of action of light both in the light growth response and in phototropism.

     

Cell wall pH and auxin transport velocity

According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport.     

A saturable site responsible for polar transport of indole-3-acetic acid in sections of maize coleoptiles

The velocity of transport and shape of a pulse of radioactive indole-3-acetic acid (IAA) applied to a section of maize (Zea mays L.) coleoptile depends strongly on the concentration of nonradioactive auxin in which the section has been incubated before, during, and after the radioactive pulse. A pulse of [³H]IAA disperses slowly in sections incubated in buffer (pH 6) alone; but when 0.5–5 M IAA is included, the pulse achieves its maximum velocity of about 2 cm h^-1. At still higher IAA concentrations in the medium, a transition occurs from a discrete, downwardly migrating pulse to a slowly advancing profile. Specificity of IAA in the latter effect is indicated by the observation that benzoic acid, which is taken up to an even greater extent than IAA, does not inhibit movement of [³H]IAA. These results fully substantiate the hypothesis that auxin transport consists of a saturable flux of auxin anions (A^-) in parallel with a nonsaturable flux of undissociated IAA (HA), with both fluxes operating down their respective concentration gradients. When the anion site saturates, the movement of [³H]IAA is nonpolar and dominated by the diffusion of HA. Saturating polar transport also results in greater cellular accumulation of auxin, indicating that the same site mediates the cellular efflux of A^-. The transport inhibitors napthylphthalamic acid and 2,3,5-triiodobenzoic acid specifically block the polar A^- component of auxin transport without affecting the nonsaturable component. The transport can be saturated at any point during its passage through the section, indicating that the carriers are distributed throughout the tissue, most likely in the plasmalemma of each cell.Abbreviations A^- auxin anion - HA undissociated auxin - IAA indole-3-acetic acid - NPA N-1-napthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

Morphogenesis in selaginella: auxin transport in the stem

Selaginella willdenovii Baker is a prostrate vascular cryptogam with a dorsiventral stem. At each major branching of the stem apex a dorsal and a ventral angle meristem is formed. The ventral meristem becomes determined as a root, and the dorsal meristem as a shoot. The present investigation examined the distribution and transport of ¹⁴C-indoleacetic acid through stem tissues as a basis for the pattern of meristem determination. Externally applied indoleacetic acid is transported into receiver blocks with a velocity of 12 millimeters per hour. Much of the auxin becomes immobilized in the tissue and is not transported. The polar ratio of auxin transport is approximately 2. Auxin is transported equally on the dorsal and the ventral sides of the stem axis, and the auxin flux in vascular tissue is twice that in the cortex. In the branch junctions twice as much auxin is transported on the dorsal side as on the ventral side, and this is held to be the consequence of the lateral branch vascular tissue connecting with the dorsal and median, but not with the ventral vascular strand of the stem axis.     

Auxin Transport as Related to Leaf Abscission during Water Stress in Cotton

Plant water deficits reduced the basipetal transport of auxin in cotyledonary petiole sections taken from cotton (Gossypium hirsutum L.) seedings. A pulse-labeling technique was employed to eliminate complications of uptake or exit of ¹⁴C-indoleacetic acid from the tissue. The transport capacity or the relative amount of radioactivity in a 30-minute pulse which was basipetally translocated was approximately 30% per hour in petioles excised from well watered seedlings (plant water potentials of approximately -4 to -8 bars). No cotyledonary leaf abscission took place in well watered seedlings. Plant water potentials from -8 to -12 bars reduced the transport capacity from 30 to 15% per hour, and although the leaves were wilted, cotyledonary abscission did not increase appreciably at these levels of stress. The threshold water potential sufficient to induce leaf abscission was approximately -13 bars and abscission increased with increasing stress while the auxin transport capacity of the petioles remained relatively constant (15% per hour). The basipetal transport capacity of well watered petioles tested under anaerobic conditions and acropetal transport tested under all conditions were typically less than basipetal transport under the most severe stress conditions. Cotyledonary abscission took place during and 24 hours after relief of stress with little or no abscission taking place 48 hours after relief of stress. Although the water potential returned to -4 bars within hours after rewatering the stressed plants, partial recovery of the basipetal transport capacity of the petioles was not apparent until 48 hours after rewatering, and at least 72 hours was required to return the transport capacity to near normal values. These data support the view that decreased levels of auxin reaching the abscission zone from the leaf blade influence the abscission process and further suggest that the length of time that the auxin supply is maximally reduced is more critical than the degree of reduction.     

Seasonal variations in the polar-transport pathways and retention sites of [3H]indole-3-acetic acid in young branches ofFagus sylvatica L.

Branches were cut from young beeches (Fagus sylvatica L.) at various stages of the annual cycle and [³H]indole-3-acetic acid (0.35 nmol) was applied to the whole surface of the apical section of each branch, just below the apical bud. The labelled pulse (moving auxin) and the following weakly radioactive zone (auxin and metabolites retained by the tissues) were localized by counting: microautoradiographss were made using cross sections from these two regions. During the second fortnight of April, auxin was transported by nearly all the cells of the young primary shoot, but the label was more concentrated in the vascular bundles. Auxin transport became the more localized: the cortical parenchyma appeared to lose its ability to transport the hormone (end of April), followed in turn by the pith parenchyma (May). Polar auxin movement at that time was limited to the outer part of the bundle (cambial zone and phloem) and to the inner part (protoxylem parenchyma). Later protoxylem parenchyma ceased to carry auxin. During the whole period of cambial activity, auxin was transported and retained mainly by the cambial zone and its recent derivatives. In September, before the onset of dormancy, and in February, at the end of the resting period, the transport pathways and retention sites for auxin were mainly in the phloem, where sieve tubes often completely lacked radiolabel. When cambial reactivation occurred in the one-year shoot, auxin was mainly carried and retained again in the cambial zone and differentiating derivatives.Abbreviation IAA indole-3-acetic acid     

1-N-naphthylphthalamic acid and 2,3,5-triiodobenzoic acid

Summary Auxin transport in corn coleoptile sections was inhibited by 2,3,5-triiodobenzoic acid (TIBA) as well as by 1-N-naphthylphthalamic acid (NPA); this inhibition was effected within 1 min of application.A particulate cell fraction-presumably plasma-membrane vesicles-specifically binds NPA and properties of these binding sites were studied using ³H-NPA and a pelletting technique. The saturation kinetics of the physiological NPA effect, i.e. the inhibition of auxin transport, is similar to that of the specific in-vitro NPA binding. Half saturation of the inhibitory effect was found with about 5×10^-7 M TIBA and with 10^-7 M NPA. Both substances also decreased the speed of movement of auxin pulses within coleoptile sections.NPA dissociates from its binding site when the particulate cell material is centrifuged through an NPA-free cushion. The NPA that is washed from its binding site can be used in another binding test without any apparent change and is chromatographically unaltered. Therefore, the NPA binding is probably reversible and non-covalent. Inhibition of auxin transport by TIBA or NPA could also be reversed when the coleoptile sections were washed in buffer.The movement of ¹³¹I-TIBA in corn coleoptiles appears to be polar in a basipetal direction. Higher concentrations of indoleacetic acid or TIBA inhibited this polar movement, suggesting that TIBA moves in the same channels as auxin. With ³H-NPA, however, no polar transport could be detected. Together with the in-vitro binding results, these data indicate that TIBA acts directly at the auxin receptor while NPA has a different receptor site.The effect of TIBA and NPA on elongation, with or without auxin, is neglegible in comparison to their effects on auxin transport.     

Does Auxin Play a Role in the Release of Apical Dominance by Shoot Inversion in Ipomoea nil?

According to the auxin-inhibition hypothesis of apical dominance,apically produced auxin moves down the stem and inhibits axillarybud outgrowth, either directly or indirectly. This hypothesishas been examined further by monitoring changes in basipetalauxin transport and endogenous auxin concentration in Ipomoeanil caused by shoot inversion, a stimulus that releases apicaldominance. The results indicate that inversion reduces auxintransport in the main stem. In upright shoots of intact plants,a 16-h pretreatment with [³H]IAA 4 cm below the apex resultsin downward movement of label and accumulation in nodes, especiallythe cotyledonary node. Label does not accumulate in the lateralbuds. GC-MS determinations of endogenous free auxin level inthe fourth node, where a lateral bud grows out following inversionof the upper part of the shoot, show no changes at 3 and 8 hafter inversion, the range of times for inversion-induced budrelease, or at 24 h, when bud outgrowth is continuing. However,inversion did cause a just-detectable decrease (approx. 10%)in the IAA level of the shoot's elongation region. Althoughauxin transport in segments of the main stem is partially inhibitedby inversion over a period shorter than the latent time of budrelease, thus providing a means for the expected depletion ofauxin in the fourth node, no depletion could be detected there.These results suggest that either a decrease in IAA level inthe main stem is not causal of bud release or that the decreasedIAA pool responsible for bud release is compartmented and cannotbe measured in whole-tissue extracts.Copyright 1993, 1999 AcademicPress Apical dominance, auxin content, auxin transport, axillary bud release, GC-MS, Ipomoea nil, Pharbitis nil, shoot inversion     

Effect of Inversion on Growth and Movement of Indole-3-acetic Acid in Coleoptiles

The effect of a 180° displacement from the normal vertical orientation on longitudinal growth and on the acropetal and basipetal movement of ¹⁴C-IAA was investigated in Avena sativa L. and Zea mays L. coleoptile sections. Inversion inhibits growth in intact sections (apex not removed) and in decapitated sections supplied apically with donor blocks containing auxin. Under aerobic conditions, inversion inhibits basipetal auxin movement and promotes acropetal auxin movement, whereas under anaerobic conditions, it does not influence the movement of auxin in either direction. Inversion retards the basipetal movement of the peak of a 30-minute pulse of auxin in corn.

The inversion-induced inhibition of basipetal auxin movement is not explained by an effect of gravity on production, uptake, destruction, exit from sections, retention in tissue, or purely physical movement of auxin. It is concluded that inversion (a) inhibits basipetal transport, the component of auxin movement that is metabolically dependent, and as a result (b) inhibits growth and (c) promotes acropetal auxin movement.

     

The Promotion of the Immobilization of Auxin in Avena Coleoptiles by Triiodobenzoie Acid

A study was made of the inhibition of auxin transport in Avena coleopliles by 2, 3, 5-triiodobenzoic acid (TIBA), using carboxyl labelled ¹⁴C indole-3-acetic acid (IAA). A transport period of 1 hour followed by an export period of 2 hours was used routinely. Treatment with TIBA resulted in increased radioactivity remaining in the coleoptile sections after the export period. This radioactivity was distributed throughout the length of the coleoptile. The increase in radioactivity was shown to be due to an increase in the amount of IAA immobilized. The action of TIBA in inhibiting auxin transport is achieved by the promotion of the immobilization of IAA.     

The transport of indole-3-acetic Acid in boron- and calcium-deficient sunflower hypocotyl segments

Transfer of sunflower (Helianthus annuus L. cv Russian Mammoth) seedlings from complete nutrient solution to solutions deficient in either boron or calcium resulted in a steady decline in the rate of auxin transport, compared to seedlings that remained in the complete solution. In seedlings transferred to solutions deficient in both B and Ca, the decline in auxin transport was greater than seedlings deficient in only one element. The transfer of B- or Ca-deficient seedlings back to the complete solution prevented further decline in auxin transport, but auxin transport did not increase to the same level as seedlings maintained in complete solution. The significant reduction in auxin transport during the early stages of B or Ca deficiency was not related to (a) reduced growth rate of the hypocotyl, (b) increased acropetal movement of auxin, or (c) lack of respiratory substrates in the hypocotyl. In addition, no difference was found in the water-extractable total and ionic Ca in B-deficient and control nondeficient hypocotyls, indicating a direct effect of B on auxin transport, rather than indirectly by affecting Ca absorption. The rate of auxin transport in hypocotyls deficient in either B or Ca, was inversely correlated with K⁺ leakage and rate of respiration. The data presented strongly support the view that there are separate sites for B and Ca in the basipetal transport of the plant hormone indoleacetic acid.     

Effects of ethylene on auxin transport

The effect of ethylene on the uptake, distribution and polar transport of C¹⁴ from indole-3-acetic acid-2-C¹⁴ and naphthalene acetic acid-1-C¹⁴ in tissue sections was studied. Test species were cotton (Gossypium hirsutum, L.) and cowpea (Vigna sinensis, Endl.). Generally, incubation of tissue or intact plants with ethylene reduced the degree of polar auxin transport. Ethylene inhibited the movement of both auxins in stem tissue and IAA in petiole tissue of cotton. The effect of ethylene on auxin movement in cow-peas was more complex. Ethylene apparently inhibited transport in younger petiole and stem tissue, but stimulated the process to a small but significant degree in basal petiole segments.

Ethylene, in some experiments, reduced C¹⁴ (auxin) uptake. This reduction was consistently smaller than the inhibition of transport. Effects upon transport were observed when uptake was not different. Differences in uptake declined as the period of incubation with auxin was lengthened, but transport was inhibited for up to 23 hours.

It is proposed that ethylene may, through its effect on transport, cause localized shortages and surpluses of auxin which in turn contribute to symptoms now associated with the response of sensitive species to ethylene.

     

Auxin transport: a new synthetic inhibitor

The new synthetic plant growth regulator DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo [5,1-a] isoindol-8-one) was examined for its effects on auxin transport. At a concentration of 0.5 mm in the receiver agar cylinders DPX1840 significantly inhibited the basipetal transport of naphthaleneacetic acid-1-¹⁴C in stem sections of Vigna sinensis Endl., Pisum sativum L., Phaseolus vulgaris L., Glycine max L., Helianthus annuus L., Gossypium hirsutum L., and Zea mays L. without significantly reducing total auxin uptake or recovery. The time sequence of the effect varied with the plant species. A similar inhibition of the basipetal movement of indoleacetic acid-1-¹⁴C was observed in intact seedlings of Phaseolus vulgaris L. In contrast to basipetal auxin transport DPX1840 had no significant effect on the acropetal movement of indoleacetic acid-1-¹⁴C in stem sections of Gossypium hirsutum L. Qualitatively the effect of DPX1840 on basipetal auxin transport was similar to that of other known auxin transport inhibitors. Quantitative differences, however, suggested the following order of activity: Naptalam>morphactin[unk]DPX1840>2,3,5-triiodobenzoic acid.     

Inhibition of Polar Auxin Transport by Ethylene

Applied ethylene influences the growth of etiolated pea stem sections cut from untreated plants, but has no effect on (14)C-indoleacetic acid uptake, polar transport or destruction. However, the capacity of the polar auxin transport system is markedly reduced in sections cut from plants grown in ethylene, while the velocity of auxin transport is unchanged under these conditions. Inhibition of the polar transport system by ethylene could underlie certain responses in which the gas produces symptoms of auxin deficiency.     

Auxin activity of substituted benzoic acids and their effect on polar auxin transport

Six dichloro-, 3 trichloro-, 2 triiodo-, and 3 heterosubstituted benzoic acids (amiben, dinoben, dicamba), and N-1-naphthylphthalamic acid have been tested for effects on growth and on polar auxin transport. Growth activity with and without kinetin was measured by effects on fresh and dry weights of 30-day cultures of fresh tobacco pith. Transport inhibition was measured by following uptake and output of IAA-2-¹⁴C through 10 mm bean epicotyl sections. The distribution of callus growth on vascularized tobacco stem segments was also observed. Avena first internode extension assays established the relative activities: dicamba > amiben > dinoben suggested by pith growth results. Growth effects of active compounds were similar with and without kinetin, except that amiben was less active with kinetin, while 2,3,6-trichlorobenzoic acid was more active with kinetin than alone. The weak auxin activity of NPA was confirmed. Transport experiments showed that NPA was the most inhibitory compound tested, followed by TIBA. Other compounds tested were at least 300 times less inhibitory to IAA transport. The best growth promoters were the least inhibitory to transport, and the most effective transport inhibitors were at best poor auxins. It is suggested that the weak auxin and auxin synergistic activity of TIBA (and perhaps 2,3-dichlorobenzoic acid) in extension growth tests arises from its inhibition of transport of endogenous or added auxin out of the sections, rather than from its intrinsic auxin activity. Chemically induced apolar callus growth on vascularized tobacco stem explants can arise from inhibition of native auxin transport, apolar growth stimulation by auxinic action of the test compound, or both.