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.     

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 level of phytohormones in monoecious and gynoecious cucumbers as affected by photoperiod and ethephon

The endogenous levels of auxin, gibberellin, and inhibitors were followed in monoecious and gynoecious cucumber (Cucumis sativus L.) plants, and in plants treated with the ethylene-releasing compound Ethephon (2-chloroethyl phosphonic acid). Higher auxin inhibitor and lower gibberellin levels were associated with female tendency. The endogenous level of gibberellin and auxin decreased in Ethephon-treated plants. Application of Ethephon induced a rise in abscisic acid. Root application of abscisic acid promoted female tendency of gynoecious cucumbers grown under conditions which increase maleness. High CO₂ levels, which are known to antagonize ethylene, increased maleness of gynoecious cucumbers. The possibility of interrelationship between gibberellin, auxin, ethylene, and abscisic acid on sex expression are discussed.     

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.     

1-Aminocyclopropane-1-Carboxylic Acid Transported from Roots to Shoots Promotes Leaf Abscission in Cleopatra Mandarin (Citrus reshni Hort. ex Tan.) Seedlings Rehydrated after Water Stress

The effect of water stress and subsequent rehydration on 1-aminocyclopropane-1-carboxylic acid (ACC) content, ACC synthase activity, ethylene production, and leaf abscission was studied in Cleopatra mandarin (Citrus reshni Hort. ex Tan.) seedlings. Leaf abscission occurred when drought-stressed plants were allowed to rehydrate, whereas no abscission was observed in plants under water stress conditions. In roots of water-stressed plants, a high ACC accumulation and an increase in ACC synthase activity were observed. Neither increase in ACC content nor significant ethylene production were detected in leaves of water-stressed plants. After rehydration, a sharp rise in ACC content and ethylene production was observed in leaves of water-stressed plants. Content of ACC in xylem fluid was 10-fold higher in plants rehydrated for 2 h after water stress than in nonstressed plants. Leaf abscission induced by rehydration after drought stress was inhibited when roots or shoots were treated before water stress with aminooxyacetic acid (AOA, inhibitor of ACC synthase) or cobalt ion (inhibitor of ethylene-forming enzyme), respectively. However, AOA treatments to shoots did not suppress leaf abscission. The data indicate that water stress promotes ACC synthesis in roots of Cleopatra mandarin seedlings. Rehydration of plants results in ACC transport to the shoots, where it is oxidized to ethylene. Subsequently, this ethylene induces leaf abscission.     

Movement and Endogenous Levels of Abscisic Acid during Water-Stress-induced Abscission in Cotton Seedlings

In an effort to investigate possible involvement of abscisic acid (ABA) in foliar abscission processes, its movement and endogenous levels were examined in cotyledons taken from cotton seedlings (Gossypium hirsutum L.) subjected to varying degrees of water deficit, a condition which initiates leaf abscission. Using a pulse-labeling technique to avoid complications of uptake and exit from the tissue, ABA-1-¹⁴C movement was observed in both basipetal and acropetal directions in cotyledonary petioles taken from well watered, stressed, and rewatered plants. The label distribution patterns obtained after 1 and 3 hours of transport under all situations of water supply were diffusive in nature and did not change when tested under anaerobic conditions. The transport capacity of the petioles ranged from 3.6 to 14.4% ABA-1-¹⁴C transported per hour at estimated velocities of 0 to 2 millimeters per hour. Comparison of basipetal and acropetal movement indicated a lack of polarity under all conditions tested. These low transport capacities and slow velocities of movement, when compared to the active transport systems associated with auxin movement, as well as the lack of anaerobic effects and polarity, suggest that ABA movement in cotton cotyledonary petiole sections is facilitated by passive diffusion. Increases in free and bound ABA in the lamina with increased water stress did not correlate with patterns of cotyledonary abscission. Thus, no evidence was found to suggest that ABA is directly involved in stress-induced abscission processes.     

Abscission: the initial effect of ethylene is in the leaf blade

The leaf blade of cotton (Gossypium hirsutum L. cv. Stoneville 213) was investigated as the initial site of ethylene action in abscission. Ethylene applied at 14 μl/l to intact 3-week-old plants caused abscission of the third true leaf within 3 days. However, keeping only the leaf blade of this leaf in air during ethylene treatment of the rest of the plant completely prevented its abscission for up to 7 days. This inhibition of abscission was apparently the result of continued auxin production in the blade since (a) the application of an auxin transport inhibitor to the petiole of the air-treated leaf blade restored ethylene sensitivity to the leaf in terms of abscission; (b) repeated applications of naphthaleneacetic acid to the leaf blade of the third true leaf, when the entire plant was exposed to ethylene, had the same preventive effect on abscission of this leaf as keeping its leaf blade in air; and (c) the inhibitory effect of ethylene on auxin transport in the petiole, which is reduced by auxin treatment, was also reduced by placing the leaf blade in air.     

Effect of Protein Synthesis Inhibitors, Auxin, and Gibberellic Acid on Abscission

Chloramphenicol, actinomycin D, and other inhibitors of protein synthesis promote abscission in several plant genera. Abscission is accelerated in species where an abscission layer is present, as well as in tissue where no abscission layer develops prior to abscission. The inhibitors promote abscission in species where cell division is reported to precede the separation processes as well as in tissues where no cell division is associated with the initiation of abscission. Indoleacetic acid (IAA) or auxin precursors, when applied with chloramphenicol and aclinomycin D, overcome the promotive effects of the inhibitors on abscission. These inhibitors apparently do not promote abscission through their effects on auxin precursor conversion, IAA transport, and IAA destruction in the petiole. IAA increases the incorporation of leucine-1-¹⁴C into a trichloroacetic acid precipitable fraction of the abscission zone under conditions where abscission is retarded. A low concentration of IAA which accelerates abscission, decreases incorporation of leucine into protein. Other promoters of abscission — chloramphenicol, d-aspartic acid, and gibberellic acid —also decrease the incorporation of leucine into the protein of the abscission zone. The data indicate that enzymes required for the degradative processes associated with abscission are already present in the abscission zone whereas a continuous synthesis of protein is required for the retention of the leaf.     

Effect of calcium, (2-chloroethyl) phosphonic acid and ethylene on bean leaf abscission

Summary The effects of CaCl₂, (2-chloroethyl) phosphonic acid (Ethephon) and ethylene on leaf abscission of debladed and intact bean plants (Phaseolus vulgaris L.) were studied. Ethephon (1000 g/l) and ethylene (8 l/l) induced abscission in debladed and intact plants in 24–72 h whereas IAA (10^-5M), cycloheximide (10^-5M) and CaCl₂ (0.068M) delayed abscission in debladed plants. CaCl₂ completely inhibited the abscission-enhancing effect of Ethephon in intact bean leaves. When CaCl₂ and Ethephon were applied simultaneously to separate halves of the leaf blade, leaves with Ethephon applied closest to the pulvinus abscised rapidly; when CaCl₂ was applied closest to the pulvinus, abscission was prevented. Calcium pre-treatment prior to ethylene (8 l/l) treatment of debladed plants delayed abscission as compared to those treated with ethylene alone.Michigan Agricultural Experiment Station Journal Article No. 6299.     

Calcium distribution in the abscission zone of bean leaves: electron microprobe x-ray analysis

The calcium content and distribution across the abscission zones of (2-chloroethyl) phosphonic acid-treated bean (Phaseolus vulgaris L. var. Contender) leaves were lower and not uniformly distributed as compared to the control. Calcium chloride-treated bean leaves had a higher calcium content, with more calcium localized in the potential abscission layer. Ethephon treatment promoted abscission in both debladed and nondebladed plants; there was a corresponding decrease in calcium in the abscission zone just prior to separation. Deblading of bean leaves under a calcium solution increased the calcium level in the abscission zone and delayed abscission.     

Effect of Ethephon, Indole Butyric Acid, and Treatment Solution pH on Rooting and on Ethylene Levels within Mung Bean Cuttings

Light-grown mung bean (Phaseolus aureus Roxb.) cuttings were treated with buffered and nonbuffered solutions of Ethephon, indole butyric acid (IBA), and the combination of both. Ethephon treatment resulted in increased tissue ethylene levels with increasing solution pH, but had no effect on rooting. IBA treatment had no effect on tissue ethylene levels, but strongly promoted rooting. Combinations of Ethephon and IBA had no effect on rooting of mung bean cuttings beyond that obtained by IBA alone.     

Sites of auxin action: regulation of geotropism, growth, and ethylene production by inhibitors of auxin transport

The inhibitors of auxin transport-NPA (N-1-naphthylphthalamic acid), DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-a] isoindol-8-one), and TIBA (2,3,5-triiodobenzoic acid)-inhibited geotropism in roots of intact Pisum sativum L. seedlings. NPA and DPX1840 also caused cellular swelling in the roots. The swelling was due to a greater inhibition of elongation than increase in weight and looked identical to the one caused by ethylene. However, ethylene did not act as an intermediate in the action of auxin transport inhibitors because all three failed to stimulate ethylene production and some of their growth-inhibiting effect was retained in the presence of saturating levels of ethylene. In the presence of 10 mum indoleacetic acid the growth-inhibiting effect of auxin transport inhibitors was lost after 18 hours. On the other hand, auxin transport inhibitors did not interfere with the ability of auxin to promote ethylene production. Growth inhibition caused by auxin transport inhibitors was reversible. Pea root sections resumed normal growth following flushing of treated sections with inhibitor-free solutions. Experiments with (14)C-2, 4-dichlorophenoxyacetic acid revealed that the herbicide and auxin transport inhibitors may have the same binding site. It was concluded that a class of structurally dissimilar compounds may share a similar physiological role since they all appear to compete with endogenous auxin for certain binding sites and they all have similar growth-regulating activities.     

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.     

Gibberellin Effect on Tryptophan Metabolism, Auxin Destruction, and Abscission in Coleus

Application of gibberellic acid (GA) to the apical region of the stem enhances ¹⁴CO₂ release from tryptophan-l-¹⁴C in cell free preparations of the apical region. Although GA when applied to the apical region markedly accelerates abscission rates of debladed petioles at the 4th node, the enhancement effect on tryptophan metabolism appears to be restricted to the apical bud region. The increased levels of diffusible auxin in Coleus stems, observed earlier by Muir and Valdovinos (1965), appear to be due to the GA effect on auxin precursor conversion rather than to an altered rate of auxin destruction. GA pre-treatment does not significantly alter destruction rates of auxin in the stem tissue. This is demonstrated by the release of ¹⁴CO₂ from IAA-1-¹⁴C by sections of internode tissue. While a multiple deblading pattern retards abscission of debladed petioles considerably, application of GA to debladed petioles at the basal region of the stem restores the normal rates of abscission at debladed distal nodes. No significant change in the abscission rates at treated nodes is observed. The GA effect on abscission at distal nodes is attributed to the effect of the growth substance on auxin precursor conversion in the apical region. In these experiments, as in the case of plants treated in the apical region with GA, auxin destruction rates in the stem are not altered significantly.     

Ethylene Evolution following Treatment with 1-Aminocyclopropane-1-carboxylic Acid and Ethephon in an in Vitro Olive Shoot System in Relation to Leaf Abscission

1-Aminocyclopropane-1-carboxylic acid (ACC) supplied via the cut base of detached olive shoots caused a burst of ethylene from leaves, but other cyclopropanes tested did not exhibit this effect. Ethephon (ET) and another ethylene-releasing compound caused a prolonged increase in ethylene evolution. ACC had only a very limited effect on leaf abscission regardless of concentration, whereas shoots placed with cut bases in ET for 60 to 80 minutes exhibited 100% leaf abscission within 90 hours. Shoots with inflorescences treated with ET just prior to anthesis began to wilt in vitro within 20 to 30 hours and failed to exhibit leaf abscission. At earlier stages of development, ET induced more leaf abscission on reproductive shoots than on vegetative shoots. It is suggested that the duration of ethylene evolution from the leaves governs their potential for abscission and that bursts of ethylene evolution even though large in amount may not induce abscission.     

Convergent evolution of shoots in land plants: lack of auxin polar transport in moss shoots

SUMMARY The shoot is a repeated structure made up of stems and leaves and is the basic body plan in land plants. Vascular plants form a shoot in the diploid generation, whereas nonvascular plants such as mosses form a shoot in the haploid generation. It is not clear whether all land plants use similar molecular mechanisms in shoot development or how the genetic networks for shoot development evolved. The control of auxin distribution, especially by polar auxin transport, is essential for shoot development in flowering plants. We did not detect polar auxin transport in the gametophytic shoots of several mosses, but did detect it in the sporophytes of mosses without shoot structure. Treatment with auxin transport inhibitors resulted in abnormal embryo development, as in flowering plants, but did not cause any morphological changes in the haploid shoots. We fused the soybean auxin-inducible promoter GH3 with a GUS reporter gene and used it to indirectly detect auxin distribution in the moss Physcomitrella patens . An auxin transport inhibitor NPA did not cause any changes in the putative distribution of auxin in the haploid shoot. These results indicate that polar auxin transport is not involved in haploid shoot development in mosses and that shoots in vascular plants and mosses are most likely regulated differently during development.     

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.     

Auxin-induced Fruit Set in Capsicum annuum L. Requires Downstream Gibberellin Biosynthesis

A hierarchical scheme for the central role of the plant hormones auxin and gibberellins in fruit set and development has been established for the model plants Arabidopsis and tomato. In the fruit crop Capsicum annuum, the importance of auxin as an early signal in fruit set has also been recognized; however, the effect of gibberellins and their interaction with auxin has not yet been studied. The aim of this study was to determine the role of gibberellin and the hierarchy between auxin and gibberellin. We applied gibberellin alone or in combination with auxin or with the gibberellin biosynthesis inhibitor paclobutrazol on stigmas of emasculated flowers. Gibberellin application enhanced fruit set, whereas application of paclobutrazol reduced fruit set. The effect of paclobutrazol treatment could be counteracted by coapplication of gibberellin but not by auxin_. These results indicate that in C. annuum, like in Arabidopsis and tomato, auxin is the major inducer of fruit set that acts in part by inducing gibberellin biosynthesis. Interestingly, gibberellin does not significantly contribute to the final fruit size but seems to play an important role in preventing flower and fruit abscission, a major determinant of production loss in C. annuum. At the same time, gibberellin together with auxin seems to balance cell division and cell expansion during fruit growth.     

Inhibition of auxin transport by isoquinolinedione herbicides

2-(p-carbethoxyphenyl)-1,3(2H,4H)-isoquinolinedione (CEPIQ), an experimental herbicide, caused effects on geotropism, which are often indicative of an effect on auxin transport, in a whole plant herbicidal screen. However, it showed little or no activity in an in vitro binding assay in corn coleoptiles for the auxin-transport inhibitor,N-1-naphthylphthalamic acid (NPA). Other active isoquinolinedione analogues of this compound did, however, exhibit significant in vitro activity. Direct measurements of auxin transport in corn coleoptiles were undertaken in an attempt to resolve the apparent discrepancy between herbicidal and binding activities. In all cases examined, compounds that were highly active on whole plants were good inhibitors of auxin transport, and compounds that were weak as herbicides showed little or no effect on auxin transport. Therefore, it is concluded that the mode of action of these isoquinolinedione herbicides is the inhibition of auxin transport. Ring-opened analogues of several isoquinolinediones were synthesized and assayed in both the transport and binding assays, in order to test whether compounds in this class express their herbicidal activity by undergoing ring-opening in vivo, yielding products that are more straightforward analogues of NPA with free carboxyl groups. The homophthalamic acids had little or no activity in both assays. On the other hand, thep-ethyl- andp-ethoxy-phenyl phthalamic acids showed auxin transport inhibition comparable to the parent isoquinolinediones, but with markedly increased binding activity. These results support the possible role of ring-opening in the generation of biological activity. However, thep-carbethoxyphenyl phthalamic acid, analogous to CEPIQ, was very weak in both assays. Thus, ring-opening in vivo cannot alone account for the biological activity of this class of compounds.