LOCALIZATION OF DEHYDROGENASE AND ACID PHOSPHATASE IN THE ABSCISSION ZONE OF BEAN LEAVES

Leaf abscission in Phaseolus vulgaris L. cv. ‘Contender’ is associated with enzymatic changes during and prior to separation. Deblading resulted in a localized increase in dehydrogenase and acid phosphatase in the abscission zone. Increased enzyme activities were observed 24–48 hr after deblading. In debladed plants separation was complete in 6–8 days. At separation, dehydrogenase activity appeared to decrease and localization was specific to the protective layer, while the petiole side had no activity. In contrast, acid phosphatase activity was observed in some layers of cells on the petiole side after separation. Ethylene treatment promoted abscission and separation occurred in 24–48 hr in both debladed and intact plants. No protective layer was formed during ethylene-induced abscission. Enzymatic changes similar to those observed in debladed control plants were observed with ethylene treatment. Ethylene induced an additional abscission layer between the pulvinus and petiole, where an abscission layer normally does not form. In this ethylene-induced abscission layer, similar enzyme activities were detected.     

A HISTOCHEMICAL STUDY OF ABSCISSION LAYER FORMATION IN THE BEAN

In debladed bean petioles calcium and dry weight increased in the abscission zone during an induction period of 14 hr. Before the microscopic appearance of the abscission layer calcium decreased in the abscission zone and increased in the petiole. Dry matter began to decrease in both the abscission zone and the petiole 24 hr after deblading. The first visual change in the cells of the abscission zone was a swelling of the pectic materials of the cell walls. This was followed by breakdown of other cell wall components, i.e., non-cellulosic polysaccharides and cellulose. The cellulose of the cell walls adjacent and distal to the abscission layer was found to be altered; however, no lignin was present during abscission layer development. The alteration of pectic materials, coupled with breakdown of cell wall components, resulted in the collapse of cells of the abscission layer just prior to separation. Auxin delayed abscission and also delayed the initial increase in calcium, the movement of calcium from the abscission zone to the petiole, and the decrease in dry weight.     

Auxin and gibberellin effects on cell growth and starch during abscission in cotton

An increase in starch content of cells in the abscission zone of the cotton explant appeared correlated with an increase in number of cells. A large increase in the number of cells in the abscission zone, concomitant with an increase in starch content, followed treatment with gibberellin as compared to auxin. In the final stages of abscission starch was hydrolyzed in the cells of the separation layer. Some starch remained after the petiole abscised.

A positive phloroglucinol-hydrochloric acid reaction in the cells of the petiole distal to the line of separation indicated the presence, not of lignin, but of soluble sugars and uronic acids. This reaction was especially intense following gibberellic acid treatment.

It was concluded that gibberellin in accelerating abscission leads to (1) an increase in cell number and starch content in the abscission zone, (2) the hydrolysis of starch in the separation layer just before abscission, and (3) the breakdown of polysaccharides and the release of soluble sugars and uronic acids. Auxin, an abscission retardant, either delays or prevents these events.

     

Studies on leaflet abscission of blue lupin leaves

Summary In blue lupin leaves, each leaflet abscises at an abscission zone situated in the pulvinus at its base. The time to abscission of leaflets of detached leaves is proportional to leaf age. Light accelerates abscission; within certain limits the acceleration is the greater the younger the leaf. At a given concentration, kinetin applied to a single leaflet accelerates leaflet abscission in young leaves kept in darkness, delays it in older ones. There is an interaction between kinetin and light which is dependent also on leaf age and kinetin concentration. The leaf can be considered as consisting of three regions, the petiole, the pulvinar region and the leaflets. The effects of kinetin and of light as well as their interactions depent on the regions of the leaf treated with these agents. Kinetin applied to a leaflet of a young leaf kept in darkness accelerates abscission, but kinetin applied to the pulvinar region of a similar leaf kept in darkness delays abscission. When any part of a leaf is illuminated, abscission is accelerated. The most light-sensitive region of the leaf is the pulvinar region, despite its relatively small area. Acceleration of abscission by light is greatest when illumination of the pulvinar region is combined with illumination of either the leaflets or the petiole. The interaction of light with kinetin is complex. Where the illuminated area includes the pulvinar region, kinetin delays abscission. This effect is most marked in the case where the pulvinar region alone is illuminated and kinetin is applied to a leaflet.Intrafoliar abscission as found in lupin leaves permits study of complex interactions of both distal and proximal stimuli involved in 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.     

EFFECTS OF INDOLE-3-ACETIC ACID AND PARACHLOROPHENOXY-ISOBUTYRIC ACID ON ABSCISSION IN PETIOLES OF DEBLADED LEAVES OF PHASEOLUS VULGARIS

The effects of indole-3-acetic acid (IAA) and p-chlorophenoxyisobutyric acid (PCIB) on rates of abscission layer formation and abscission were investigated. The primary leaves of Phaseolus vulgaris were used as test material. Treatment at the distal end of one petiole of the pair from debladed primary leaves with 1% IAA inhibited the abscission of that petiole and accelerated the abscission of its opposite untreated partner. PCIB applied simultaneously with IAA counteracted the accelerating effect of IAA on the opposite untreated petiole. This influence increased with increasing concentrations of PCIB. Anatomical studies revealed that PCIB, although it counteracted the effect of IAA on the rate of abscission, had no effect on abscission layer formation. In other words abscission layer formation takes place under the influence of the auxin despite the presence of the antiauxin. The centripetal sequence of abscission layer formation was found in all cases.     

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.     

Anatomical Changes and Immunolocalization of Cellulase during Abscission as Observed on Nitrocellulose Tissue Prints

A fundamental event in abscission is the breakdown of cell wall material in a discrete zone of cells known as the separation layer. Three dimensional images produced by viewing tissue prints of abscission zones on nitrocellulose (NC) membranes with incident illumination showed changes in the tissue integrity taking place in the separation layer as the process of abscission proceeded. The cell softening which occurs due to the dissolution of the cell wall appeared in the tissue prints as a diffuse line at the anatomical transition between the pulvinus and petiole and was easily observed on NC tissue prints of either longitudinal or serial cross-sections through abscission zones. In bean leaf abscission the dissolution of cell walls has been correlated with the appearance of a form of cellulase with an isoelectric point of pH 9.5. Antibodies specific for this enzyme were used to study the localization of 9.5 cellulase in the distal abscission zone of Phaseolus vulgaris L., cv Red Kidney after tissue printing on NC. It was found that 9.5 cellulase was localized in the separation layer but also occurred in the vascular tissue of the adjacent pulvinus. No antibody binding was observed in nonabscising tissue or preimmune controls. These results confirm previous biochemical studies and demonstrate that immunostaining of nitrocellulose tissue prints is a fast and reliable method to localize proteins or enzymes in plant tissue.     

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.     

Breakdown of middle lamella pectin by ●OH during rapid abscission in Azolla

Azolla, a small water fern, abscises its roots and branches within 30 min upon treatment with various stresses. This study was conducted to test whether, in the rapid abscission that occurs in Azolla, breakdown of wall components of abscission zone cells by ^●OH is involved. Experimentally generated ^●OH caused the rapid separation of abscission zone cells from detached roots and the rapid shedding of roots from whole plants. Electron microscopic observations revealed that ^●OH rapidly and selectively dissolved a well‐developed middle lamella between abscission zone cells and resultantly caused rapid cell separation and shedding. Treatment of abscission zones of Impatiens leaf petiole with ^●OH also accelerated the separation of abscission zone cells. However, compared with that of Azolla roots, accelerative effects in Impatiens were weak. A large amount of ^●OH was cytochemically detected in abscission zone cells both of Azolla roots and of Impatiens leaf petioles. These results suggest that ^●OH is involved in the cell separation process not only in the rapid abscission in Azolla but also in the abscission of Impatiens. However, for rapid abscission to occur, a well‐developed middle lamella, a unique structure, which is sensitive to the attack of ^●OH, might be needed.     

STYLE ABSCISSION IN THE CITRON (CITRUS MEDICA L.) AND OTHER CITRUS SPECIES: MORPHOLOGY,PHYSIOLOGY, AND CHEMICAL CONTROL WITH PICLORAM

Style abscission was studied in detail in citrons (Citrus medica L.) and other citrus varieties. The course of style abscission was followed under orchard conditions and also in an “explant” system consisting of pistils implanted in an agar-sucrose medium and maintained at 25 C in a humid chamber. Morphological and anatomical observations carried out with the explant system revealed a prominent swelling of cell layers proximal to the separation layer prior to abscission. Tests with explants from flowers of different developmental stages showed that before the end of anthesis only the ovaries are capable of performing abscission while style abscission is possible only at a later stage, presumably after fertilization had occurred. Ethylene was able to induce ovary abscission at later stages but could not induce earlier style abscission. Picloram (4, amino-3,5,6-trichloropicolinic acid) increased the percentage of style persistence in citron varieties which naturally tend to retain their styles. Picloram also induced style persistence in Valencia oranges and Eureka lemons, which naturally show 100 % style abscission. Hormonal determinations showed that the style had higher levels of auxin than the ovary but also higher levels of inhibitors, which increased towards the time of style 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.     

Anatomical and ultrastructural changes associated with ethylene-induced defoliation of guayule explants

Explants of guayule,Parthenium argentatum Gray, were treated with concentrations of ethephon varying from 0.5 to 10gl ^-1 for six days. The cut ends of the shoots were immersed in the solutions and allowed to stand so that the ethylene-releasing agent entered the explant via the transpiration stream. With higher concentrations of ethephon, defoliation of the explants commenced after one day and was 100% effective after six days. Lower concentrations were less effective. Examination of the petiole bases of treated explants at the light microscope level revealed enhanced development of abscission layers and hydrolytic degradation of the tissue immediately distal to these layers. This led to separation of the leaf which had become senescent. Food reserves appeared to have been mobilised from the senescent leaves. Histochemical staining and ultrastructural observations indicated loss of insoluble polysaccharides and cellulose from the induced separation layer. Pectic substances were lost to a lesser extent.The financial support of Cooperative Scientific Programmes of the C.S.I.R. and technical assistance of the Electron Microscope Unit of the University of Natal, Pietermaritzburg is gratefully acknowledged.     

STEM ABSCISSION IN THE TUMBLEWEED,PSORALEA

Abscission was studied in Psoralea argophylla Pursh, a prairie tumbleweed. This process is responsible for the detachment of the stem from its perennial base. In a single plant abscission may proceed through one or more intercalary meristems located at or near ground level. During senescence a separation layer differentiates within each of these meristems. Chemical changes occur within the compound middle lamellae which result in the conversion of insoluble pectic compounds into soluble forms. Cell separation takes place only after removal of the pectic compounds, meristematic activity, and attenuation of cell walls. Unlike reports of other angiosperms the protective layer is not formed adjacent to the separation layer, but forms some distance underground at the stem-root junction.     

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     

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.     

Histochemical changes in the developing abscission layer in fruits of Prunus cerasus L.

Summary Abscission layer formation in the sour cherry (Prunus cerasus L.) during fruit maturation occurred in the transition zone between the fruit and the pedicel. The abscission layer, consisting of 5–8 rows of cells, was first identified by its low affinity for haematoxylin. The walls of cells in the abscission layer contained less total polysaccharides than adjacent cells. The pectins were degraded and the cellulose was partially broken down resulting in cell separation. The Ca level in the abscission zone decreased and Ca and Mg were lost from the walls of cells in the layer during abscission. After the abscission layer formed, cells associated with the layer had a lower capacity to bind ⁴⁵Ca than cells distal or proximal to the layer.Michigan Agricultural Experiment Station Journal Article No. 4607     

GRAFTING AND ROOTING OF LEAVES OF GUAREA (MELIACEAE): EXPERIMENTAL STUDIES ON LEAF AUTONOMY

The pinnately compound, indeterminate leaves of G. glabra and G. guidonia were air layered, detached from their original shoots, and grown on their own adventitious root systems for up to 58 mo and 26 mo, respectively. The detached leaves grew in the same indeterminate manner and reached sizes similar to attached leaves. Although detached leaves grew autonomously, they never produced shoot buds. Leaves of both species were grafted onto their own stems and cut free of their original leaf bases. Leaf scions survived and grew for up to 29 mo and 20 mo, respectively, similar to ungrafted leaves. Axillary branches were grafted onto subtending leaves. Branch scions grew on their leaf stocks for over 30 mo and 24 mo, respectively, after being cut free from the branch bases. Secondary growth of the leaf axis (petiole) was promoted, and vascular tissues of leaf and branch axes were continuous. However, the unlignified basal region of the leaf, including the abscission zone, remained unchanged after grafting. The results indicate that proximity of roots and bypassing the abscission zone did not enhance leaf longevity or pinna production. The presence of a growing branch on a leaf did not modify the structure of the abscission zone, which suggests that the zone is strongly committed or developmentally fixed.     

The Role of Cell Expansion in the Abscission of Impatiens sultani leaves

The histological events occurring during the latter stages ofabscission were followed continuously in longitudinal slicesthrough the petiole base of Impatiens sultani Hook. It appearsthat the middle lamella of the cortical parenchyma cells isdegraded first. This is followed by an expansion of these cellsand a concomitant stretching and separation of the collenchymaand vascular trace. The parenchyma cells continue to enlargeuntil they become virtually spherical, a process which finallyruptures the last restraining xylem vessels. The increased volumeof the parenchyma cells appears to be principally due to a conformationalchange in cell shape from a near regular hexagonal prism toa sphere of similar surface area. The dimensions of the prismaticcells are such that most of them change into spheres whose diametersare the same as the transverse distance between the oppositesides of the six axially orientated faces of the prisms. Cellularexpansion is thus entirely directed along the axis of the petiole.The significance of these observations to the general anatomyand mechanism of fracture of abscission zones is discussed. Impatiens sultani Hook., abscission, cell expansion, cell wall degradation, cell shape