FORMATION OF CALLOSE AND LIGNIN DURING LEAF ABSCISSION

Deblading of bean leaves promoted the formation of callose and lignin in the abscission zone. The abscission layer became evident three days after deblading. The greatest increase in callose occurred in about two layers of cells during the development of the abscission layer. Four days after deblading, only a few layers of cells on the distal side of the abscission layer showed an increase in lignin. Lignification continued to expand to 8–10 layers of cells at the time of separation. After separation, the lignified cells remained with the petiole. Sieve elements in the abscission zone were covered with callose plugs and the vessels were occluded with tyloses.     

ABSCISIN,AUXIN, AND GIBBERELLIN EFFECTS ON THE DEVELOPMENTAL ASPECTS OF ABSCISSION IN COTTON (GOSSYPIUM HIRSUTUM)

The effects of accelerating and retarding amounts of abscisin (Ab II), auxin (IAA), and gibberellin (GA₃) on abscission in explants of 14-day-old cotton (Gossypium hirsutum L.) seedlings were studied. Applications of Ab II, a potent accelerant (0.025 μg/abscission zone), resulted in a lysigenous breakdown of cells in a weakly defined separation layer in contrast to GA₃, an accelerant (0.01 μg/abscission zone), and IAA, a retardant (0.125 μg/abscission zone), which resulted in a schizogenous type of breakdown of cells in a well-defined separation layer, three or more rows of cells wide. Separation usually commenced adaxially with GA₃, abaxially with IAA and in the controls, and either ad- or abaxially with Ab II. Cell division preceded abscission, the number of cells increasing greatly within 24 hr after GA₃ treatment. Tyloses formed in vessel elements throughout the explant, both distal and proximal to the plane of separation in all treatments and in the controls. The retardant, IAA, appeared to stimulate tyloses formation. Tylosis development was not causal but was secondarily related to abscission.     

Mechanism of Action of Abscission Accelerators

Abscission zone explants of Gossypium hirsutum L., Cassia fistula L., and Coleus blumei Benth. were used to investigate correlations between endogenous rates of ethylene evolution and time of abscission. Additions of 0.1 nl/ml ethylene to the explants markedly accelerated abscission; continuous aeration of the explants, to prevent accumulation of small amounts of endogenously produced ethylene, inhibited abscission compared with that of sealed controls. Substances that stimulated abscission simultaneously accelerated ethylene evolution on all three species and at any position of application. The positional effects of auxin are explained as being due to differences in transport in the explant. Thus, distally applied auxin inhibits abscission, regardless of the accelerated rate of ethylene evolution, by being rapidly transported to the abscission zone. Auxin applied proximally stimulates abscission because it is unable to move as rapidly to the abscission zone and the ethylene effect becomes dominant. Ethylene was found to be most effective on aged tissues, and it is concluded that abscission rates are determined by an increase in sensitivity of the tissue to the ethylene that is already being produced.     

Methyl jasmonate induces the formation of secondary abscission zone in stem of Bryophyllum calycinum Salisb

Methyl jasmonate (JA-Me) at a concentration of 0.5 % induced the formation of secondary abscission zone and senescence in several types of stem explants (only internode segment, internode segment with nodes and without leaves, internode segment with nodes and debladed petioles) of Bryophyllum calycinum when it was applied in various places of the stem or the debladed petiole as lanolin paste. In the presence of small leaves in stem explants methyl jasmonate also induced the formation of secondary abscission zone and senescence but the presence of larger leaves completely inhibited methyl jasmonate-induced processes. Auxin, (indole-3-acetic acid, IAA), at a concentration of 0.1 % extremely prevented the formation of secondary abscission zones and senescence in the stem tissues induced by methyl jasmonate. Similar relationship between auxin and methyl jasmonate to induce the formation of secondary abscission zone and senescence was found in decapitated shoot of the intact plant. Mechanisms of the formation of secondary abscission zone are also discussed in terms of the interaction of methyl jasmonate with auxin.     

Further examination of abscission zone cells as ethylene target cells in higher plants

BACKGROUND AND AIMS: Two aspects of the competence of abscission zone cells as a specific class of hormone target cell are examined. The first is the competence of these target cells to respond to a remote stele-generated signal, and whether ethylene acts in concert with this signal to initiate abscission of the primary leaf in Phaseolus vulgaris. The second is to extend the concept of dual control of abscission cell competence. Can the concept of developmental memory that is retained by abscission cell of Phaseolus vulgaris post-separation in terms of the inductive/repressive control of beta-1,4-glucan endohydrolase (cellulase) activity exerted by ethylene/auxin be extended to the rachis abscission zone cells of Sambucus nigra? METHODS: Abscission assays were performed using the leaf petiole-pulvinus explants of P. vulgaris with the distal pulvinus stele removed. These (-stele) explants do not separate when treated with ethylene and require a stele-generated signal from the distal pulvinus for separation at the leaf petiole-pulvinis abscission zone. Using these explants, the role of ethylene was examined, using the ethylene action blocker, 1-methyl cyclopropene, as well as the significance of the tissue from which the stele signal originates. Further, leaf rachis abscission explants were excised from the compound leaves of S. nigra, and changes in the activity of cellulase in response to added ethylene and auxin post-separation was examined. KEY RESULTS: The use of (-stele) explants has confirmed that ethylene, with the stele-generated signal, is essential for abscission. Neither ethylene alone nor the stelar signal alone is sufficient. Further, in addition to the leaf pulvinus distal to the abscission zone, mid-rib tissue that is excised from senescent or green mid-rib tissue can also generate a competent stelar signal. Experiments with rachis abscission explants of S. nigra have shown that auxin, when added to cells post-separation can retard cellulase activity, with activity re-established with subsequent ethylene treatment. CONCLUSIONS: The triggers that initiate and regulate the separation process are complex with, in bean leaves at least, the generation of a signal (or signals) from remote tissues, in concert with ethylene, a requisite part of the process. Once evoked, abscission cells maintain a developmental memory such that the induction/repression mediated by ethylene/auxin that is observed prior to separation is also retained by the cells post-separation.     

The positional differentiation of ethylene-responsive cells in rachis abscission zones in leaves of Sambucus nigra and their growth and ultrastructural changes at senescence and separation

Summary In leaves of S. nigra, fragmentation of the rachis follows the autumnal abscission of leaflets and the high levels of ethylene produced by the senescing blades. Fragmentation is accompanied by cell growth and ultrastructural changes in a zone of cells precisely differentiated at the separation zone. Studies with explants from the rachis show that those that contain an abscission zone increase in freshweight by as much as 50% before and during cell separation. Cell growth changes are induced by ethylene but not by auxin, and are restricted to explants that contain the separation zone cells. In ethylene, enlarging cells of the zone show cytoplasmic activation indicated by dilated dictyosomes, enhanced production of Golgi vesicles, elongated profiles of rough endoplasmic reticulum, a crenellated plasmalemma, and the apparent discharge and accumulation of cytoplasmic vesicles within the desmotubules of the branched plasmodesmata. Degradation of the middle lamella and cell wall matrix could be associated with the release of hydrolytic enzymes on the disruption of the vesicles. Although ultrastructural changes of a similar but limited nature occur in all cells of the rachis in response to ethylene, only those that are morphologically delimited as zone cells exhibit the growth and separation that leads to rachis fragmentation. It is proposed that abscission can occur only at the sites of the positional differentiation of these special ethylene-responsive target cells.Abbreviation IAA indole-acetic acid (auxin)     

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.     

The positional differentiation of abscission zones during the development of leaves of Sambucus nigra and the response of the cells to auxin and ethylene

Summary Abscission in the leaf rachis of Sambucus nigra L. is preceded by a positional differentiation of zone cells that enlarge and separate in response to ethylene but not to auxin. These cells are absent from youngest leaves, and such leaves do not abscind even in ethylene; other cells of the immature rachii will enlarge in response to auxin. These two classes of target cells are always recognisable by their opposing responses to auxin and ethylene. Prior to separation zone cells exposed to ethylene show considerable activation of the cytoplasm, many polysomes, elongate endoplasmic reticulum and highly dilated dictyosomes with many associated vesicles. Treatment with auxin precludes these changes, and abscission is always retarded: high levels of ethylene must be added to overcome the auxin inhibition. The differentiation of zone cells and their ethylene-stimulated growth and activation are prerequisites for rachis abscission in Sambucus. Such cell development may be of general occurrence prior to organ abscission in plants.Abbreviation IAA indole-3yl-acetic acid     

Deposition of callose in relation to abscission of citrus leaves

When leaves of Citrus sinensis (L.) Osbeck cv. Shamouti senesce, they become more susceptible to abscission and the proximal 2 mm of their lamina-petiole abscission zones exhibit callose deposition. The degree of senescence, assayed with the DAR-WIN image processor (Telewski et al. 1983), was positively correlated in a linear fashion with callose deposition. Explant of non-senescing leaves were observed. Excision of the leaf at the stem-petiole junction induced callose deposition throughout the petiole, but not in the lamina. Callose deposition began immediately upon excision and reached a maximum at 3 h. It then decreased slightly and remained at the same level for up to 5 days. Exogenous compounds that decrease callose deposition, e.g. laminarase and 2-deoxy-D-glucose, inhibited the rate of abscission of explants. Compounds that promote callose deposition, e.g. uridine diphosphoglucose and mannose, increased the rate of abscission of explants. Exogenous callose, e.g. laminarin, increased the rate of abscission. It is not known how callose might be causally involved in promoting abscission.     

Induced Abscission Sites in Internodal Explants of Impatiens sultani: A New System for Studying Positional Control: with an Appendix: A Mathematical Model for Abscission Sites

Intemodes from Impatiens sultani shoots, explanted into sterileculture, often developed a transverse separation layer afterone to two weeks and the top then abscised from the bottom ofthe explant. Such abscission occurred more rapidly and in agreater proportion of explants when 00001 per cent auxin (IAA)was provided basally and when younger intemodes and shorterexplants were used. The distance of the separation layer fromthe base of the explant varied little with explant length, butincreased with the concentration of auxin applied basally. It seems that in this adventitious abscission the processesof positional definition and differentiation proceed withoutpause, whereas in normal abscission the position is definedearly in development but the final stage of differentiationof the separation layer is delayed until much later when theorgan senesces. To account for the results from the internodal explants andfrom surgical operations on shoots as well as for the characteristicposition of abscission sites of leaves and fruits, we suggestthat the position of abscission is controlled primarily by auxinacting as a morphogen: abscission sites occur at Y-junctionsjust above the base of the arm with the lower activity and auxinstatus, or in single axes above a region of higher auxin status.In both sites, the auxin concentration decreases in the apicaldirection. This hypothesis is supported by a mathematical model (see Appendix)of the interaction of diffusive and polar transport in controllingthe concentration gradient along intemodes with specified auxinconcentrations maintained basally. The model allows predictionsconcerning the site and timing of abscission which accord withobservations on intemodal explants. Impatiens sultani Hook., abscission, auxin, differentiation, diffusion coefficient, IAA, morphogen, polar transport coefficient, positional control, separation layer     

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.     

Some ultrastructural observations on the nature of foliar abscission in Impatiens sultani

Summary Both scanning and transmission electron microscopes have been used to study the anatomy of the abscission zone of Impatiens sultani Hook. Evidence is presented to show that the fracture line follows the middle lamella in all the living cells of the abscission zone including those in the vascular traces. The separation of these cells is preceded by a breakdown of the middle lamellar region of the wall. The characteristics of this process vary in different cell types. Accompanying this breakdown is an enlargement of inner cortex cells mainly in a direction parallel to the axis of the petiole. It is suggested that this expansion of cells is necessary to produce the tensions which rupture the cuticle and xylem vessels prior to separation. The occurrence of transfer cells and tyloses in the abscission zone is also described and the physiological implications of their presence discussed.     

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.     

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     

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     

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.     

Temporal and Spatial Distribution of Auxin Response Factor Genes During Tomato Flower Abscission

Abscission facilitates growth and reproduction and improves plant defenses against pathogens. This tightly regulated process is triggered by environmental cues and hormones such as ethylene and auxin. Because auxin is crucial for abscission, auxin response factors (ARFs) may play important roles in this process. Here, we examined changes in gene expression during abscission in tomato, focusing on regulation of genes encoding ARFs. Specifically, we analyzed the pattern of ARF gene expression in tomato flower pedicel explants treated with ethylene, the ethylene blocker 1-methylcyclopropene (1-MCP), or auxin to determine how auxin and ethylene affect ARF gene expression. In addition, we examined the spatial and temporal distribution of IAA during abscission by examining transgenic tomato plants expressing an IAA-inducible promoter fused to the GUS reporter gene (the P5::GUS ‘Chico III’ line). Flower removal from the explants quickly induced abscission by ethylene, which was inhibited by exogenous auxin or 1-MCP. During early abscission, auxin (or 1-MCP) regulated the expression of various ARFs, including ARF1, 2, 3, 4, 5, 7, 8-1, 9, 11, 12, 13, 13-1, 14, and 17, whereas ethylene had the opposite effect on most of these genes. Further analysis shows that during this stage, auxin may mediate the expression of ARF8-1, 9, 11, 12, 13, 13-1, and 14, whereas ethylene may mediate ARF13-1. During the later stage of abscission, ARF2, 8, 10, 11, and 19 were upregulated, and 8-1, 12, 13, and 13-1 were downregulated, compared with nonabscising parts of plants. Fluorometric GUS analysis indicated that GUS activity in the abscission zone remained stable at 4 h and sharply decreased after 8 h until abscission was complete (32 h).     

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.     

Effect of Ethylene on Peroxidase Activity

Ethylene increased the peroxidase activity of nine out of ten varieties of sweet potato (Ipomoea batatas (L.) Lam.) root disks tested. The increase which was observed four hours after ethylene treatment was partially overcome by carbon dioxide. The increase was inhibited by actinomycin D and cycloheximide, indicating de novo protein synthesis. Electrophoretic separation on polyacrylamide gels indicated the appearance of two new peroxidase bands. Peroxidase activity in bean petiole explants was localized around the separation layer. Ethylene caused a small increase in peroxidase activity in the petiolar portion of the explant. Phenolic substances had no effect on abscission consistent with their proposed roles as cofactors for auxinoxidase, indicating that auxin-oxidase does not play a role in abscission of Coleus blumei Benth. abscission zone explants.