Abscission: the role of ethylene modification of auxin transport

The role of ethylene-mediated reduction of auxin transport in natural and ethylene-induced leaf abscission was studied in the cotton (Gossypium hirsutum L., cv. Stoneville 213) cotyledonary leaf system. The threshold level of ethylene required to cause abscission of intact leaves was between 0.08 and 1 μl/l with abscission generally occurring 12 to 24 hours following ethylene fumigation. The threshold level of ethylene required to reduce the auxin transport capacity in the cotyle-donary petiole paralleled that required for stimulation of abscission. In plants where cotyledons are allowed to senesce naturally there is a decline in auxin transport capacity of petioles and increase in ethylene synthesis of cotyledons. The visible senescence process which precedes abscission requires up to 11 days, and increases in ethylene production rates and internal levels were detected well before abscission. Ethylene production rates for entire cotyledons rose to 2.5 mμ1 g⁻¹ hr⁻¹ and internal levels of 0.7 μl/l were observed. These levels appear to be high enough to cause the observed decline in auxin transport capacity. These findings, along with those of others, indicate that ethylene has several roles in abscission control (e.g., transport modification, enzyme induction, enzyme secretion). The data indicate that ethylene modification of auxin transport participates in both natural abscission and abscission hastened by exogenous ethylene.     

Starch Synthetase, Phosphorylase, ADPglucose Pyrophosphorylase, and UDPglucose Pyrophosphorylase in Developing Maize Kernels

Three types of whole plant experiments are presented to substantiate the concept that an important function of ethylene in abscission is to reduce the transport of auxin from the leaf to the abscission zone. (a) The inhibitory effect of ethylene on auxin transport, like ethylene-stimulated abscission, persists only as long as the gas is continuously present. Cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants placed in 14 μl/l of ethylene for 24 or 48 hours showed an increase in leaf abscission and a reduced capacity to transport auxin; but when returned to air, auxin transport gradually increased and abscission ceased. (b) Ethylene-induced abscission and auxin transport inhibition show similar sensitivities to temperature. A 24-hour exposure of cotton plants to 14 μl/l of ethylene at 8 C resulted in no abscission and no significant inhibition of auxin transport. Increasing the temperature during ethylene treatment resulted in a progressively greater reduction in auxin transport with abscission occurring at [unk]27 C where auxin transport was inhibited over 70%. (c) Auxin pretreatment reduced both ethylene-induced abscission and auxin transport inhibition. No abscission occurred, and auxin transport was inhibited only 18% in cotton plants which were pretreated with 250 mg/l of naphthalene acetic acid and then placed in 14 μl/l of ethylene for 24 hours. In contrast, over 30% abscission occurred, and auxin transport was inhibited 58% in the corresponding control plants.     

Effect of ethylene on auxin transport and metabolism in midrib sections in relation to leaf abscission of woody plants

Abstract The relationship between ethylene-induced leaf abscission and ethylene-induced inhibition of auxin transport in midrib sections of the leaf blade of Citrus sinensis L. Osbeck, Populus deltoides Bart, and Eucalyptus camaldulensis Dehn. was studied. These species differed greatly in their abscission response to ethylene. The kinetic trend of abscission resembled that of the inhibition of auxin transport in all three species. It is suggested that one of the main actions of ethylene in the leaf blade is to inhibit auxin transport in the veinal tissues, thus reducing the amount of auxin transported from the leaf blade to the abscission zone. Ethylene inhibited transport of both IAA (indole-3-acetic acid) and NAA (α-naphthaleneacetic acid) in the midrib sections. However, while ethylene enhanced the conjugation of IAA with aspartic acid and glucose in the apical (absorbing) segment of the midrib sections, it had little effect on the conjugation of NAA. The data indicate that auxin destruction through conjugation does not play a major role in the inhibition of auxin transport by ethylene.     

Abscission: potentiating action of auxin transport inhibitors

Reduction in petiolar auxin transport has been proposed as one of the functional actions of endogenous or exogenous ethylene as it regulates intact leaf abscission. If this hypothesis is correct, auxin-transport inhibitors should hasten the rate or amount of abscission achieved with a given level of ethylene. Evidence presented here indicates that the hypothesis is correct. Three auxin transport inhibitors promoted ethylene-induced intact leaf abscission when applied to specific petioles or the entire cotton plant (Gossypium hirsutum L., cv. Stoneville 213). In addition, the transport inhibitors caused rapid abscission of leaves which usually do not abscise under the conditions employed. No stimulation of abscission occurred during the initial 3 to 5 days after plants were treated with transport inhibitors unless such treatments were coupled with exogenous ethylene or that derived from 2-chloroethylphosphonic acid. However, vegetative cotton plants did abscise some of their youngest true leaves during the 2nd and 3rd weeks of exposure to transport inhibitor alone. Taken as a whole, the results indicate that reducing the auxin supply to the abscission zone materially increases sensitivity to ethylene, a condition which favors a role of endogenous ethylene in abscission regulation. Such a role of ethylene indicates the importance of auxin-ethylene interactions in the over-all hormone balance of plants and specific tissues.     

Ethylene-induced leaf abscission in cotton seedlings : the physiological bases for age-dependent differences in sensitivity

The speed of ethylene-induced leaf abscission in cotton (Gossypium hirsutum L. cv LG-102) seedlings is dependent on leaf position (i.e. physiological age). Fumigation of intact seedlings for 18 hours with 10 microliters per liter of ethylene resulted in 40% abscission of the still-expanding third true (3°) leaves but had no effect on the fully expanded first true (1°) leaves. After 42 hours of fumigation with 50 microliters per liter of ethylene, total abscission of the 3° leaves occurred while <50% abscission of the 1° leaves was observed. On a leaf basis, endogenous levels of free IAA in 1° leaves were approximately twice those of 3° leaves. Free IAA levels were reduced equally (approximately 55%) in both leaf types after 18 hours of ethylene (10 microliters per liter) treatment. Ethylene treatment of intact seedlings inhibited the basipetal movement of [¹⁴C]IAA in petiole segments isolated from both leaf types in a dose-dependent manner. The auxin transport inhibitor N-1-naphthylphthalamic acid increased the rate and extent of ethylene-induced leaf abscission at both leaf positions but did not alter the relative pattern of abscission. Abscission-zone explants prepared from 3° leaves abscised faster than 1° leaf explants when exposed to ethylene. Ethyleneinduced abscission of 3° explants was not appreciably inhibited by exogenous IAA while 1° explants exhibited a pronounced and protracted inhibition. The synthetic auxins 2,4-D and 1-naphthaleneacetic acid completely inhibited ethylene-induced abscission of both 1° and 3° explants for 40 hours. It is proposed that the differential abscission response of cotton seedling leaves is primarily a result of the limited abscission-inhibiting effects of IAA in the abscission zone of the younger leaves.     

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.     

The stimulating effects of ethylene and auxin on petiole elongation and on hyponastic curvature are independent processes in submerged Rumex palustris

The flooding-tolerant plant species Rumex palustris (Sm.) responds to complete submergence with stimulation of petiole elongation mediated by the gaseous hormone ethylene. We examined the involvement of auxin in petiole elongation. The manipulation of petiolar auxin levels by removing the leaf blade, or by addition of synthetic auxins or auxin transport inhibitors, led to the finding that auxin plays an important role in submergence-induced petiole elongation in R. palustris. A detailed kinetic analysis revealed a transient effect of removing the auxin source (leaf blade), explaining why earlier studies in which less frequent measurements were taken failed to identify any role for auxin in petiole elongation. We previously showed that the onset of stimulated petiole elongation depends on a more upright petiole angle being reached by means of hyponastic (upward) curvature, a differential growth process that is also regulated by ethylene and auxin. This raised the possibility that both ethylene and auxin stimulate elongation only indirectly by influencing hyponastic growth. We show here that the action of ethylene and auxin in promoting petiole elongation in submerged R. palustris is independent of the promoting effect that these hormones also exert on the hyponastic curvature of the same petiole.     

Auxin transport and the regulation of petiole abscission in cotton (Gossypium hirsutum L.): A comparison of indol-3yl-acetic acid and phenylacetic acid

Distal applications of indol-3yl-acetic acid (IAA) to debladed cotyledonary petioles of cotton (Gossypium hirsutum L.) seedlings greatly delayed petiole abscission, but similar applications of phenylacetic acid (PAA) slightly accelerated abscission compared with untreated controls. Both compounds prevented abscission for at least 91 h when applied directly to the abscission zone at the base of the petiole. The contrasting effects of distal IAA and PAA on abscission were correlated with their polar transport behaviour-[1-¹⁴C]IAA underwent typical polar (basipetal) transport through isolated 30 mm petiole segments, but only a weak diffusive movement of [1-¹⁴C]PAA occurred.Removal of the shoot tip substantially delayed abscission of subtending debladed cotyledonary petioles. The promotive effect of the shoot tip on petiole abscission could be replaced in decapitated shoots by applications of either IAA or PAA to the cut surface of the stem. Following the application of [1-¹⁴C]IAA or [1-¹⁴C]PAA to the cut surface of decapitated shoots, only IAA was transported basipetally through the stem. Proximal applications of either compound stimulated the acropetal transport of [¹⁴C]sucrose applied to a subtending intact cotyledonary leaf and caused label to accumulate at the shoot tip. However, PAA was considerably less active than IAA in this response.It is concluded that whilst the inhibition of petiole abscission by distal auxin is mediated by effects of auxin in cells of the abscission zone itself, the promotion of abscission by the shoot tip (or by proximal exogenous auxin) is a remote effect which does not require basipetal auxin transport to the abscission zone. Possible mechanisms to explain this indirect effect of proximal auxin on abscission are discussed.     

Abscission in Coleus: Light and Phytohormone Control

Light control of leaf abscission in Coleus (Coleus blumei Benthcv. Ball 2719 Red) appears to be regulated by the quantity ofendogenous auxin transported from the leaf blade to the abscissionzone. Gas chromatographic—mass spectrophotometric analysisindicated that diffusate collected from leaf tissue treatedwith red light contained significantly higher levels of auxinthan dark and far-red light-treated leaf tissue. In addition,diffusate from red light-treated tissue inhibited abscissionof leafless petioles while diffusate from far-red light-treatedtissue promoted abcission when compared with diffusate fromdark-treated tissue. The effect of red light on abscission couldbe mimicked by IAA, but not by other phytohormones. An auxintransport inhibitor, 2, 3, 5-triiodobenzoic acid (TIBA), appliedeither as a lanolin ring around the petiole or vacuum infiltratedinto tissue, could completely eliminate any red light effecton abscission. The data are consistent with a phytochrome-mediatedlight regulation of endogenous auxin level in the leaf whichthen controls abscission. Key words: Abscission, Coleus, IAA, plant hormones, red (far-red) light, TIBA     

Citrus leaf abscission. Regulatory role of exogenous auxin and ethylene on peroxidases and endogenous growth substances

Abstract The abscission of citrus leaf explants demonstrates the well-known enhancing effect of ethylene and the delaying one of auxin when treatment is started at excision time. Total peroxidase activity increases differently in tissues of the blade, abscission zone, and petiole. The highest activity at zero time is recovered in abscission zone in which also the response to the abscission regulators is the most visible. Isoperoxidase profiles are modified in opposite directions by ethylene and auxin respectively. Both regulators affect the activity of the same cathodic and anodic isoperoxidases without any qualitative changes. By the same time, auxin-like compounds increase in isolated abscission zones at 24 h from excision and decrease at 48 h. The level of one inhibitor complex undergoes an inverse variation. It is suggested that the increase in auxin during the first stage of abscission is necessary for influencing the growth of cells which is required to cause abscission.     

Transdifferentiation of Mature Cortical Cells to Functional Abscission Cells in Bean

Abscission explants of bean (Phaseolus vulgaris L.) were treated with ethylene to induce cell separation at the primary abscission zone. After several days of further incubation of the remaining petiole in endogenously produced ethylene, the distal two-thirds of the petiole became senescent, and the remaining (proximal) portion stayed green. Cell-to-cell separation (secondary abscission) takes place precisely at the interface between the senescing yellow and the enlarging green cells. The expression of the abscission-associated isoform of β-1,4-glucanhydrolase, the activation of the Golgi apparatus, and enhanced vesicle formation occurred only in the enlarging cortical cells on the green side. These changes were indistinguishable from those that occur in normal abscission cells and confirm the conversion of the cortical cells to abscission-type cells. Secondary abscission cells were also induced by applying auxin to the exposed primary abscission surface after the pulvinus was shed, provided ethylene was added. Then, the orientation of development of green and yellow tissue was reversed; the distal tissue remained green and the proximal tissue yellowed. Nevertheless, separation still occurred at the junction between green and yellow cells and, again, it was one to two cell layers of the green side that enlarged and separated from their senescing neighbors. Evaluation of Feulgen-stained tissue establishes that, although nuclear changes occur, the conversion of the cortical cell to an abscission zone cell is a true transdifferentiation event, occurring in the absence of cell division.     

Disruption of the Polar Auxin Transport System in Cotton Seedlings following Treatment with the Defoliant Thidiazuron

The effect of the defoliant thidiazuron (TDZ) on basipetal auxin transport in petiole segments isolated from cotton (Gossypium hirsutum L. cv LG102) seedlings was examined using the donor/receiver agar block technique. Treatment of intact seedlings with TDZ at concentrations of 1 micromolar or greater resulted in a dose-dependent inhibition of ¹⁴C-IAA transport in petiole segments isolated 1 or 2 days after treatment. Using 100 micromolar TDZ, the inhibition was detectable 19 hours after treatment and was complete by 27 hours. Both leaves and petiole segments exhibited a marked increase in ethylene production following treatment with TDZ at concentrations of 0.1 micromolar or greater. The involvement of ethylene in this TDZ response was evaluated by examining the effects of two inhibitors of ethylene action: silver thiosulfate, 2,5-norbornadiene. One day after treatment, both inhibitors effectively antagonized the TDZ-induced inhibition of auxin transport. Two days after TDZ treatment both inhibitors were ineffective. The decrease in IAA transport in TDZ treated tissues was associated with increased metabolism of IAA. The transport of ¹⁴C-2,4-dichlorophenoxyacetic acid was also inhibited by TDZ treatment. This inhibition was not accompanied by increased metabolism. Incorporation of TDZ into the receiver blocks had no effect on auxin transport. The ability of the phytotropin N-1-naphthylphthalamic acid to stimulate IAA uptake from a bathing medium was reduced in TDZ-treated tissues. This reduction is thought to reflect a decline in the auxin efflux system following TDZ treatment.     

Rates of production and internal levels of ethylene in the vegetative cotton plant

The internal levels of ethylene in and the production ratesof ethylene by various parts of the cotton plant {Gossypiumhirsutum L. cultivar Acala 4–42) were determined by separatetechniques. Wounding of tissue, which resulted from both techniques,increased ethylene production. A peak in wound-induced ethyleneoccurred around the second to third hour after wounding andgradually declined until the sixth or seventh hour when ethyleneproduction began to level off. Using two techniques, with numerousobservations from each technique, ethylene levels were foundto be relatively uniform throughout the vegetative cotton plant.There was a small trend for levels of ethylene to increase fromthe bottom (oldest tissue) to the top of the plant (youngesttissue). Of major interest was the observation that the productionrates and internal concentrations of ethylene in the petiolewere two to six times higher than those in the leaf blade. Thisrelative concentration of ethylene in the petiole supports thehypothesis that ethylene is a natural regulator of abscissionwhich acts, in part, through modification of auxin transport. ¹ A contribution of die Texas Agricultural Experiment Station.Supported in part by Grant GB-5640, National Science Foundationand grants from the Cotton Producers Institute. The data forthis paper was taken from a Master of Science Thesis by J. A.M. submitted to Texas A & M University January 1970. ² Present address: Department of Agronomy, Purdue University,Lafayette, Indiana, U.S.A. (Received June 29, 1971; )     

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.

     

Leaf Age and Ethylene-induced Abscission

Ethylene has been generally credited with promoting the abscission of the oldest leaves on a plant first. Vegetative cotton (Gossypium hirsutum L.) seedlings are an exception to this generalization. Under some conditions the younger, apical, unexpanded, or partially expanded leaves abscise before the less young, basal leaves or cotyledons. The degree or extent of apical leaf abscission increases with ethylene concentration and with plant age from 2 to 5 weeks. The response is promoted by auxin transport inhibitors. Usually the leaves which abscise first are those which have just unfolded and ones apical to the opened but unexpanded leaves. With plants with eight or nine leaves and macroscopic leaf buds, after the initial loss of unexpanded leaves, abscission tends to progress downward from the youngest remaining leaves and upward from the oldest leaves. The findings indicate that some characteristic(s) of apical leaves increases their sensitivity to ethylene. The characteristic may be differences in the abscission process between expanded and unexpanded leaves or differences in the hormone complement of the different leaves. Work is under way to modify this young leaf abscission response in an effort to determine its cause.     

Temperature Sensitivity of the Latent Phase in Ethylene-induced Elongation

The temperature sensitivity is reported for the latent period preceding ethylene-induced elongation in the adaxial half of the leaf petiole of Helianthus annuus. When intact plants were exposed to 10 μl of ethylene/l of air over the temperature range 18 to 35 C, the minimum latent time was 62 minutes at 28 C and the maximum was 132 minutes at 18 C. The temperature coefficient, Q₁₀, changed from 2.1 below 28 C, to 0.7 above. In 100 μl of ethylene/l of air, the latent time was reduced by 14% at 18 C, but was significantly increased at 28 and 38 C. These results show that the latent period in the elongation response of the petiole to ethylene cannot be reduced below about 60 minutes by raising either the leaf temperature or the atmospheric ethylene concentration.     

The physiological significance of phenylacetic Acid in abscising cotton cotyledons

The physiological role of phenylacetic acid (PAA) as an endogenous regulator of cotyledon abscission was examined using cotton (Gossypium hirsutum L. cv LG 102) seedlings. Application of 100 micromolar or more PAA to leafless cotyledon abscission-zone explants resulted in the retardation of petiole abscission and a decrease in the rise of ethylene evolution that normally accompanies aging of these explants in vitro. The partial inhibition of ethylene evolution in these explants by PAA was indirect since application of this compound stimulated short-term (<24 hours) ethylene production. PAA treatment partially suppressed the stimulation of petiole abscission elicited by either ethylene or abscisic acid. Both free and an acid-labile, bound form of PAA were identified in extracts prepared from cotyledons. No discernible pattern of changes in free or bound PAA was found during the course of ethylene-induced cotyledon abscission. Unlike indole-3-acetic acid, transport of PAA in isolated petiole segments was limited and exhibited little polarity. On the whole, these results are not consistent with the direct participation of PAA in the endogenous regulation of cotyledon abscission.     

GIBBERELLIN AND AUXIN RELATIONSHIPS IN ABSCISSION

Gibberellic acid (GA) has no effect on abscission when applied proximally or distally to the abscission zones of debladed petioles of Coleus. Application of GA to the stem apex increases the rate of abscission of debladed petioles. The effect on abscission is accompanied by an increase in the level of endogenous auxin in the stem. Correspondingly proximal applications of indoleacetic acid (IAA) accelerate abscission, whereas the longevity of the debladed petiole approaches that of the intact leaf only in the presence of a continuous distal supply of IAA. No correlation is found between petiole elongation and its longevity. The experimental data support the view that auxin acts at the abscission zone in regulating separation processes and that the effect of GA is through its effect on the level of endogenous auxin.     

Petiole Growth in Ranunculus sceleratus L.: Ethylene Synthesis and Submergence

Petioles of the celery-leaved buttercup (Ranunculus sceleratusL.) elongate in response to treatment with ethylene in air whenthe leaf blades are attached. An enhanced rate of elongationgrowth also occurs when the leaves are submerged. Submergencecauses an increase in extractable ethylene gas within the tissues,and these levels appear to approach those required to saturatethe ethylene-promoted elongation growth response. Coincidentwith a rise in ethylene in the tissues is a dramatic increasein the level of I-aminocyclopropane-1-carboxylic acid (ACC),the precursor of ethylene. Both the petiole and leaf blade tissueshave a similar capacity to evolve ethylene in the presence ofadded ACC. However, in air the leaf blade evolves more ethylenefrom endogenous resources than the petiole. The simultaneousincreases in ethylene and ACC levels in submerged tissues areconsidered in terms of the low diffusivity of ethylene in water,the ‘autocatalytic’ effect of ethylene on ethylenebiosynthesis and the rôle of both carbon dioxide and oxygenfluxes in ethylene metabolism of submerged tissues. Ranunculus sceleratus, celery-leaved buttercup, petiole growth, submergence, ethylene metabolism