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.     

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: 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.     

Abscission responses to moisture stress, auxin transport inhibitors, and ethephon

The three abscission-inducing agents - water stress, Ethephon, and auxin transport inhibitors-acted synergistically to promote leaf fall in cotton (Gossypium hirsutum L.). However, the synergism was primarily between stress and Ethephon. Auxin transport inhibitors did not promote the effect of stress alone, only promoted the effect of Ethephon in well watered plants and gave a very small promotion with stress and Ethephon together. Abscission was rapid in stressed plants treated with Ethephon and an auxin transport inhibitor, while leaves fell more slowly from well watered plants treated with Ethephon alone. This suggests that water stress or auxin transport inhibitors influence initial events in abscission; since an auxin transport inhibitor will replace the effect of stress but not Ethephon, an initial event in stress-induced abscission appears to be inhibition of auxin transport. Ethephon promoted lateral bud release, and auxin transport inhibitors did not duplicate that effect alone or promote it in combination with Ethephon.     

Reduction of auxin transport capacity with age and internal water deficits in cotton petioles

Auxin transport was examined in leaf petioles taken from the upper, middle, and lower leaf canopy of large cotton plants. The ability of petioles to transport auxin decreased with age (position) of the leaves. Plant water deficit reduced transport regardless of age. These correlations support the view that reduced transport capacity of petioles plays a significant role in the induction of abscission of lower or older leaves during water deficits.     

Movement of Kinetin and Gibberellic Acid in Leaf Petioles during Water Stress-induced Abscission in Cotton

Movement of [¹⁴C]kinetin and [¹⁴C]gibberellic acid was examined in cotton (Gossypium hirsutum L.) cotyledonary petiole sections independent of label uptake or exit from the tissue. Sections 20 millimeters in length were taken from well watered, stressed, and poststressed plants. Transport capacity was determined using a pulse-chase technique. Movement of both kinetin and gibberellic acid was found to be nonpolar with a velocity of 1 millimeter per hour or less, suggesting passive diffusion. Neither water stress nor anaerobic conditions during transport of labeled material affected the transport capacity of the petioles.     

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.     

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.     

Polar transport of kinetin in tissues of radish

Polar transport of kinetin-8-¹⁴C occurred in segments of petioles, hypocotyls, and roots of radish (Raphanus sativus L.). The polarity was basipetal in petioles and hypocotyls and acropetal in roots. In segments excised from seedlings with fully expanded cotyledons, indole-3-acetic acid was required for polarity to develop. In hypocotyl segments isolated at this stage, basipetal and acropetal movements were equal during the first 12 hours of auxin treatment after which time acropetal movement declined. Pretreatment with auxin eliminated this delay in the appearance of polarity. In hypocotyl segments excised from seedlings with expanding cotyledons, exogenous auxin was unnecessary for polarity. Potassium cyanide abolished polarity at both stages of growth by allowing increased acropetal movement. The rate of accumulation of kinetin in receiver blocks was greater than the in vivo increase in cytokinin content of developing radish roots.     

The Role of Basipetal Auxin Transport in the Positional Control of Abscission Sites Induced in Impatiens sultani Stem Explants

If segments of Impatiens sultani stem are explanted and incubated,separation layers often form across them and lead to abscission.To test the suggested role of auxin concentration in controllingthe position of abscission sites, explants were labelled byapplying [¹⁴C]IAA to the shoot tip 4 h prior to explanting;transport of auxin applied in this way seems to resemble thatof endogenous auxin. During subsequent incubation of explantsfor 20 h, basipetal transport resulted in ¹⁴C accumulating justabove the base of the explants (nearly 80 % in the bottom 4mm of 24 mm explants). In internodal explants that had beenwounded at explanting by incising one side so as to sever avascular bundle, and in nodal explants with the leaf removed,the ¹⁴C also accumulated just above the wound or node to abouttwice the concentration otherwise expected; this accumulationwas probably due to basipetal transport being impeded by vasculardiscontinuity at the wound or node. Accumulation just abovethe base, or above a wound or node, resulted in gradients of¹⁴C concentration (presumably reflecting endogenous auxin concentration)decreasing in the morphologically upward direction at each ofthese three positions where abscission sites tend to occur. Impatiens sultani, abscission, auxin, IAA, node, polarized transport, positional control, separation layer, wounding     

Effects of Water Stress on the Ultrastructure of Leaf Cells of Sorghum bicolor

The subcellular changes which occurred in sorghum leaves during increasing water stress and subsequent rewatering are described. Stomata were closed, abscisic acid levels were elevated, and the amounts of starch in the bundle sheath chloroplasts were much reduced by - 14 bars leaf water potential. Swelling of the outer chloroplast membrane, and reorganization of the tonoplast to form small vesicles from the large central vacuole, occurred by a leaf water potential of - 37 bars. Complete structural disruption of the tonoplast, as previously described for maize was not found. On rewatering, large amounts of starch reappeared within three hours. These findings strengthen the hypothesis that maintenance of tonoplast integrity is an important factor in the ability of plants to withstand drought.     

Water Stress Enhances Ethylene-mediated Leaf Abscission in Cotton

Abscission of cotyledonary leaves from cotton (Gossypium hirsutum L. cv. Stoneville 213) seedlings occurred following relief from water stress. The amount of abscission was related to the magnitude of the plant water deficit. Leaf abscission promoted by exogenous ethylene was enhanced in seedlings subjected to water stress. Treatment with ethylene (2.0 to 3.2 microliters of ethylene per liter of air for 24 hours) raised the threshold plant water potential required to induce abscission from —17 to —7 bar, indicating that the stress caused the tissue to become predisposed to ethylene action. Based on the abscission response curve for seedlings treated with ethylene while under water stress, this apparent predisposition was developed as the plant water potentials reached the —7 to —10 bar range. The abscission-promoting effects of ethylene in combination with water stress were reversed with 15% CO₂ at plant water potentials above —12 bar, but the CO₂ reversal was lost at lower water potentials. These results are compatible with the concept that ethylene plays a regulatory role in leaf abscission induced by water stress.     

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.     

Involvement of ethylene in the action of the cotton defoliant thidiazuron

The effect of the defoliant thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea) on endogenous ethylene evolution and the role of endogenous ethylene in thidiazuron-mediated leaf abscission were examined in cotton (Gossypium hirsutum L. cv Stoneville 519) seedlings. Treatment of 20- to 30-day-old seedlings with thidiazuron at concentrations equal to or greater than 10 micromolar resulted in leaf abscission. At a treatment concentration of 100 micromolar, nearly total abscission of the youngest leaves was observed. Following treatment, abscission of the younger leaves commenced within 48 hours and was complete by 120 hours. A large increase in ethylene evolution from leaf blades and abscission zone explants was readily detectable within 24 hours of treatment and persisted until leaf fall. Ethylene evolution from treated leaf blades was greatest 1 day posttreatment and reached levels in excess of 600 nanoliters per gram fresh weight per hour (26.7 nanomoles per gram fresh weight per hour). The increase in ethylene evolution occurred in the absence of increased ethane evolution, altered leaf water potential, or decreased chlorophyll levels. Treatment of seedlings with inhibitors of ethylene action (silver thiosulfate, hypobaric pressure) or ethylene synthesis (aminoethoxyvinylglycine) resulted in an inhibition of thidiazuron-induced defoliation. Application of exogenous ethylene or 1-aminocyclopropane-1-carboxylic acid largely restored the thidiazuron response. The results indicate that thidiazuron-induced leaf abscission is mediated, at least in part, by an increase in endogenous ethylene evolution. However, alterations of other phytohormone systems thought to be involved in regulating leaf abscission are not excluded by these studies.     

Recovery of photosynthesis in sunflower after a period of low leaf water potential

Photosynthesis was studied in sunflower plants subjected to 1 to 2 days of desiccation and then permitted to recover. The leaf water potential to which leaves returned after rewatering was dependent on the severity of desiccation and the evaporative conditions. Under moderately evaporative conditions, leaf water potential returned to predesiccation levels after 3 to 5 hours when desiccation was slight. Leaf water potentials remained below predesiccation levels for several days after rewatering when leaf water potentials decreased to −13 to −19 bars during desiccation. Leaf water potential showed no sign of recovery when leaf water potentials decreased to −20 bars or below during desiccation. The lack of full recovery of leaf water potential was attributable to increased resistance to water transport in the roots and stem. The resistance ultimately became large enough to result in death of the leaves because net water loss continued even after the soil had been rewatered.     

Cellular and ultrastructural changes in mesophyll and bundle sheath cells of maize in response to water stress

Ultrastructural changes were correlated with leaf water potential, relative water content, and abscisic acid levels in the leaf. Mesophyll cells were more prone to damage than bundle sheath cells at a leaf water potential of -18.5 bars. Tonoplast breakdown and cell disruption occurred in 25% of the mesophyll cells. On rewatering, these disrupted cells did not recover. In bundle sheath cells, starch, lost at about -13.5 bars leaf water potential, reappeared within 2.5 hours of rewatering.     

The Role of Auxin in the Apical Regulation of Leaf Abscission in Cotton (Gossypium hirsutum L.)

The petiole abscission induced by deblading cotyledonary leavesof cotton (Gossypium hirsutum L. cv. Delta Pine) was acceleratedby the presence of the intact shoot apex or, in decapitatedplants and explants, by application to the stem (proximal application)of indol-3yl-acetic acid (IAA) or 1-aminocyclopropane-l-carboxylicacid (ACC). IAA and ACC accelerated the abscission of debladedpetioles whether applied above or below the cotyledonary node.Transport of IAA to the node was not required for the responseto proximal IAA. [2,3-¹⁴C]ACC was readily transported to thenodal region whether applied to the stem above or below thenode. Application of IAA or ACC to the stem did not induce theabscission of intact leaves or of debladed petioles treateddistally with IAA The acceleration of abscission by proximal IAA, but not thatcaused by ACC, was prevented if explants were treated with a-aminooxyaceticacid (AOA), an inhibitor of ACC-synthase. AOA also preventedthe acceleration of abscission caused by the shoot apex. Theprogress of abscission in debladed explants was greatly delayedby silver thiosulphate (STS—an inhibitor of ethylene action),whether or not the explants were treated with IAA or ACC. Itis suggested that the speeding effects of the shoot apex andof proximal auxin on the abscission of debladed petioles requiresauxin-induced ACC synthesis. The possibility is discussed thatACC may function as a mobile abscission promoter Key words: Abscission, ACC, ACC-synthase, cotton (Gossypium), proximal auxin     

Movement of Indoleacetic Acid in Coleoptiles of Avena sativa L. II. Suspension of Polarity by Total Inhibition of the Basipetal Transport

Acropetal and basipetal movement of indole-3-acetic acid through coleoptiles of Avena sativa L. was studied. Sections 10-mm long were supplied with either apical or basal sources containing C(14) carboxyl-labeled indoleacetic acid (10(-5)m). Anaerobic conditions inhibit metabolically dependent movement (transport) thus reducing basipetal but not acropetal movement. Total inhibition of basipetal transport abolishes the polarity of auxin uptake and movement. The nonpolar movement that remains in anaerobic sections is free diffusion with an average diffusion coefficient of approximately 1 x 10(-4) mm(2) per second. During an 8-hour diffusion, at least the first millimeter of the section comes to equilibrium at approximately the same concentration as the donor.Acropetal movement is probably by diffusion and is accompanied by an aerobic immobilization of indoleacetic acid that increases more than proportionally to concentration. Anaerobic conditions totally prevent this immobilization and reduce acropetal uptake but not the amount of indoleacetic acid moving into the upper parts of the section; there is, therefore, no evidence for acropetal transport.Polarity of auxin movement in aerobic coleoptile sections is achieved by strict basipetal transport of auxin. The basipetal transport may intensify the polarity by recycling auxin that is moving acropetally.     

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.     

Auxin-Responsive DR5 Promoter Coupled with Transport Assays Suggest Separate but Linked Routes of Auxin Transport during Woody Stem Development in Populus

Polar auxin transport (PAT) is a major determinant of plant morphology and internal anatomy with important roles in vascular patterning, tropic growth responses, apical dominance and phyllotactic arrangement. Woody plants present a highly complex system of vascular development in which isolated bundles of xylem and phloem gradually unite to form concentric rings of conductive tissue. We generated several transgenic lines of hybrid poplar (Populus tremula x alba) with the auxin-responsive DR5 promoter driving GUS expression in order to visualize an auxin response during the establishment of secondary growth. Distinct GUS expression in the cambial zone and developing xylem-side derivatives supports the current view of this tissue as a major stream of basipetal PAT. However, we also found novel sites of GUS expression in the primary xylem parenchyma lining the outer perimeter of the pith. Strands of primary xylem parenchyma depart the stem as a leaf trace, and showed GUS expression as long as the leaves to which they were connected remained attached (i.e., until just prior to leaf abscission). Tissue composed of primary xylem parenchyma strands contained measurable levels of free indole-3-acetic acid (IAA) and showed basipetal transport of radiolabeled auxin (³H-IAA) that was both significantly faster than diffusion and highly sensitive to the PAT inhibitor NPA. Radiolabeled auxin was also able to move between the primary xylem parenchyma in the interior of the stem and the basipetal stream in the cambial zone, an exchange that was likely mediated by ray parenchyma cells. Our results suggest that (a) channeling of leaf-derived IAA first delineates isolated strands of pre-procambial tissue but then later shifts to include basipetal transport through the rapidly expanding xylem elements, and (b) the transition from primary to secondary vascular development is gradual, with an auxin response preceding the appearance of a unified and radially-organized vascular cambium.