Separation of abscission zone cells in detached Azolla roots depends on apoplastic pH

In studies on the mechanism of cell separation during abscission, little attention has been paid to the apoplastic environment. We found that the apoplastic pH surrounding abscission zone cells in detached roots of the water fern Azolla plays a major role in cell separation. Abscission zone cells of detached Azolla roots were separated rapidly in a buffer at neutral pH and slowly in a buffer at pH below 4.0. However, cell separation rarely occurred at pH 5.0–5.5. Light and electron microscopy revealed that cell separation was caused by a degradation of the middle lamella between abscission zone cells at both pH values, neutral and below 4.0. Low temperature and papain treatment inhibited cell separation. Enzyme(s) in the cell wall of the abscission zone cells might be involved in the degradation of the pectin of the middle lamella and the resultant, pH-dependent cell separation. By contrast, in Phaseolus leaf petioles, unlike Azolla roots, cell separation was slow and increased only at acidic pH. The rapid cell separation, as observed in Azolla roots at neutral pH, did not occur. Indirect immunofluorescence microscopy, using anti-pectin monoclonal antibodies, revealed that the cell wall pectins of the abscission zone cells of Azolla roots and Phaseolus leaf petioles looked similar and changed similarly during cell separation. Thus, the pH-related differences in cell separation mechanisms of Azolla and Phaseolus might not be due to differences in cell wall pectin, but to differences in cell wall-located enzymatic activities responsible for the degradation of pectic substances. A possible enzyme system is discussed.     

Anatomy of leaf abscission in the Amur honeysuckle (Lonicera maackii, Caprifoliaceae): a scanning electron microscopy study

Lonicera maackii (Rupr.) Maxim. (Amur honeysuckle) is native to Asia and an important ornamental in China. However, the anatomy of leaf abscission (shedding) in L. maackii had not been studied previously. Such work is needed not only because knowledge of the leaf abscission process is important for a horticultural species like L. maackii but also because leaf abscission is probably the least understood abscission process, as it occurs so rapidly. Therefore, our objective was to use scanning electron microscopy (SEM) to examine the progression of leaf abscission in L. maackii at the cellular level. L. maackii branches with leaves were regularly collected in Beijing, China over the 2-month period in which leaves abscise, and examined with SEM. We found that, unlike in model species, the cortex is involved in abscission, forming an “abaxial gap.” We discovered that there is no discrete abscission zone prior to the onset of abscission and that no cell divisions precede abscission. An abscission zone did become evident well after the abscission process had begun, but its cells were enlarged, not constricted as in typical abscission zones. In the abaxial gap, intact cells separated at their middle lamella, but in the abscission zone, cell separation involved the entire wall, which is not typical. We did observe expected mechanical fission of vascular tissues. While the leaf abscission process we observed in L. maackii has similarities with model systems, aspects deviate from the expected.     

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.     

Leaf Abscission Zones in Molinia caerulea (L.) Moench, the Purple Moor Grass

Leaf separation and loss in the grass Molinia caerulea (L.)Moench was investigated using scanning electron microscopy.Leaf senescence and subsequent shedding of leaves was precededby the formation of a leaf abscission zone. The zone containscells which have more than doubled their wall thickness to greaterthan 0-4 µm. The line of fracture associated with thezone principally followed the middle lamellae, leaving intactcells on the fracture faces. Molinia should prove an interestingmodel in which to study abscission processes in the Gramineae. Molinia caerulea, leaf abscission zone, electron microscopy     

Cellulase activity and localization during induced abscission of Coleus blumei

Treatment with dimethipin (2,3-dihydro-5,6-dimethyl-1,4-dithiin 1,1,4,4 tetroxide) inhibited the increase in cellulase activity and decrease in breakstrength associated with the normal course of abscission in Coleus. Application of the surfactant UBI-1126 (Emery OAL 20 in isopropyl alcohol) increased cellulase activity and accelerated the process of abscission in Coleus expiants within 24 h of application. Cellulase activity was localized histochemically at the electron microscopic level in surfactant-treated tissue. The enzyme activity was localized primarily in the cell wall, middle lamella, and paramural bodies of abscission zone cells.     

Enzymatic and anatomical changes in abscission zone cells of apple fruits induced by Ethephon

The enhancement of fruit abscission zone formation with ethephon treatment caused an increase in soluble proteins, endo-cellulase, exo-polygalacturonase and peroxidase activities. Exo-cellulase and endo-polygalacturonase did not show any relationship with apple abscission. The separation of cells initiated in the cortex region and progressed towards vascular tissue. Cell separation in the cortex appeared to be due to dissolution of middle lamella but vascular tissues ruptured mechanically.     

The Anatomy of Fruit Abscission in Loganberries

Loganberry fruits abscise at the base of the receptacle, justdistal to the sepals. As the fruit ripens, all cells of theabscission zone expand. The central parenchyma cells, due totheir position, appear to be the driving force behind abscission.Their expansion causes early cell-separation within a superficialzone of small cells and rupture of the epidermis at the sepal/receptaclejunction without prior dissolution of cell walls. However, othercells within the abscission zone have their walls degraded,mostly in the region of the middle lamella, as ripening progresses. Xylem transfer cells are found in abundance in the vascularbundles supplying the sepals. The outward curve of these bundlesinto the sepals brings the transfer cells into close proximitywith the abscission zone. A comparison of their distributionin loganberries with that in raspberries (MacKenzie, 1979),which are closely related but abscise at a different site, suggeststhat transfer cells may be implicated in the abscission process. The inevitable structural weakness brought about by the paucityof vascular tissue in the abscission zone relative to the morerobust pedicel may also predispose this area to separation. Anatomy, abscission, loganberry, Rubus idaeus x R, ursinus, Mailing Sunberry, transfer cells structure, fruit     

The genesis of intercellular spaces in developing leaves ofPhaseolus vulgaris L.

Summary Observations by light, transmission electron and scanning electron microscopy have shown that intercellular spaces (ICS) are formed schizogenously in expanding leaves ofPhaseolus vulgaris. ICS formation occurs in predictable positions at the junctions between three or more cells, and follows three phases of development. The first, initiation, phase occurs soon after cell division, and is marked by the formation of an electron-dense osmiophilic body, probably proteinaceous, at the end of the cell plate/middle lamella of the daughter cell wall and across the adjacent piece of the primary wall of the mother cell. This part of the mother cell wall is digested, involving cellulolysis. The second phase, of cell separation, is marked by the first appearance of the ICS. InPhaseolus primary leaves this phase begins about day 3 after sowing, at which time the leaf area is about 1 cm². In the final enlargement phase, lysis of cell wall material continues in the region of the middle lamella, and mechanical tensions arising from the rapid expansion of the lamina lead to further separation of the mesophyll cells so that spaces enlarge and merge.     

Anatomy of Ethylene-induced Petal Abscission in Pelargonium x hortorum

When viewed under the light microscope, the abscission zoneat the petal base of Pelargonium x hortorum consisted of smallcells which, when stained with Toluidine Blue, possessed denselystained cells walls. After treatment with 1 µl l^-1 ethyleneat 22°C, the force required to separate the petals fromthe receptacle declined after a lag phase of only 30 min, withseparation complete 60-90 min later depending upon the stageof development of the flower. Transmission electron micrographsof the petal abscission zones showed evidence of cell wall degradation,particularly in the middle lamella. These cells also containedextensive rough endoplasmic reticulum and numerous Golgi bodiesribosomes. When abscission was complete, cells at the fractureface showed evidence of breakdown of cellular compartmentalization,often with little sign of an intact tonoplast. Scanning electronmicrographs of recently-abscissed surfaces showed that the epidermalcells surrounding the abscisson zone were turgid and rounded,whereas those of the mesophyll cells were partially collapsed.The micrographic evidence is consistent with the hypothesisthat ethylene-induced separation is caused by rapid enzymaticof the cell walls.Copyright 1993, 1999 Academic Press Abscission, cell walls, ethylene, flower, Pelargonium x hortorum     

An ultrastructural study of abscission zone cells with special reference to the mechanism of enzyme secretion

Summary The ultrastructural changes which occur during the foliar abscission ofImpatiens sultani Hook. andColeus blumei Benth. have been described. In both cases fracture of the separation zone results from a modification of the wall and cleavage along the line of the middle lamella. This process starts at the corners of the cells and in regions rich in plasmodesmata.During the period of wall breakdown, cellular integrity is maintained and the membrane degradation described by others was not observed. Plasmolysis studies confirmed that the separation zone cells retained their selective permeability characteristics until well after wall fracture. Quantitative data are presented to show that there is an increase in the frequency of rough endoplasmic reticulum, dictyosomes and dictyosome vesicles during the period when wall-hydrolyzing enzyme secretion is occurring. These changes are interpreted as reflecting an increase in demand for the secretory machinery of the endomembrane system. The possible involvement of plasmodesmata in this process is also discussed.     

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)     

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     

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     

Leaf Abscission in Soybean: Cytochemical and Ultrastructural Changes following Benzylaminopurine Treatment

6-benzylaminopurine (BAP) delays leaf abscission of soybeanGlycine max (L.) Merr. Abscission of the distal pulvinus ofprimary leaves was induced in 12-d-old seedlings or explantsby removal of the leaf blade. BAP applied to the cut end ofthe pulvinus following leaf blade removal delayed abscission.Discoloration of the pulvinus occurred before abscission commencedand the number of grana in chloroplasts within cortical parenchymacells of the pulvinus decreased over time following leaf bladeremoval. BAP prevented discoloration of pulvinus tissues anda decrease in grana number. Starch grains within amyloplastsof cells of the starch sheath in the pulvinus disappeared followingleaf blade removal, whereas starch accumulated within the abscissionzone prior to abscission. BAP prevented this apparent redistributionof starch and instead promoted an increase in starch withinplastids of cortical parenchyma cells of the pulvinus. Duringthe abscission process, cells within the separation layer enlargedand their nuclei and nucleoli became more evident prior to theirseparation from one another. Cell separation resulted from breakdownof middle lamellae and partial degradation of primary cell walls.Cycloheximide applied directly to the external surface of theabscission zone inhibited abscission in a similar way to theBAP treatment. These results suggest that BAP prevents abscissionby altering patterns of starch distribution in the pulvinusand abscission zone and by inhibiting the synthesis of proteinsthat typically appear de novo in induced abscission zone tissues. Key words: Benzylaminopurine, BAP, Soybean, Pulvinus, Abscission, amyloplast.     

Anatomical and physiological effects of silver thiosulfate on ethylene-induced abscission inColeus

Petioles of expiants ofColeus blumei Benth. exposed to 20 l/l ethylene abscised within 36 h. Pretreatment of expiants with 4 mM silver thiosulfate (STS) inhibited ethylene-induced abscission. Delaying treatment with STS reduced its effectiveness in retarding ethylene-promoted abscission, suggesting that some events leading to abscission are initiated during the first hours of ethylene treatment. Microscopic study of abscission zones of ethylene-treated expiants showed greatly increased amounts of rough endoplasmic reticulum, disruptions of the plasma membrane, and some cell separation in the region of the middle lamella. Pretreatment with STS prevented ethylene-induced reorganization of the endomembrane system and the subsequent middle lamellar dissolution.     

The SERK1 receptor‐like kinase regulates organ separation in Arabidopsis flowers

Through a sensitized screen for novel components of pathways regulating organ separation in Arabidopsis flowers, we have found that the leucine‐rich repeat receptor‐like kinase SOMATIC EMBRYOGENESIS RECEPTOR‐LIKE KINASE1 (SERK1) acts as a negative regulator of abscission. Mutations in SERK1 dominantly rescue abscission in flowers without functional NEVERSHED (NEV), an ADP‐ribosylation factor GTPase‐activating protein required for floral organ shedding. We previously reported that the organization of the Golgi apparatus and location of the trans‐Golgi network (TGN) are altered in nev mutant flowers. Disruption of SERK1 restores Golgi structure and the close association of the TGN in nev flowers, suggesting that defects in these organelles may be responsible for the block in abscission. We have also found that the abscission zones of nev serk1 flowers are enlarged compared to wild‐type. A similar phenotype was previously observed in plants constitutively expressing a putative ligand required for organ separation, INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), suggesting that signalling through IDA and its proposed receptors, HAESA and HAESA‐LIKE2, may be deregulated in nev serk1 abscission zone cells. Our studies indicate that in addition to its previously characterized roles in stamen development and brassinosteroid perception, SERK1 plays a unique role in modulating the loss of cell adhesion that occurs during organ abscission.     

Three different polygalacturonases are expressed in tomato leaf and flower abscission, each with a different temporal expression pattern.

Abscission, or organ separation, is accompanied by a marked increase in hydrolases, which are responsible for the degradation of the middle lamella and the loosening of the primary cell wall surrounding cells in the separation layer. We recently reported on the cloning of a tomato (Lycopersicon esculentum) polygalacturonase (PG) cDNA, TAPG1, expressed during leaf and flower abscission. In addition to TAPG1, we have cloned two more PG cDNAs (TAPG2 and TAPG4) that are also expressed during leaf and flower abscission. The peptide sequences for the three abscission PGs are relatively similar (76-93% identity) yet different from the those of tomato fruit PG (38-41% identity). None of the three abscission PG mRNAs are expressed in fruit, stems, petioles, or anthers of fully open flowers. An RNase protection assay revealed that all three PGs are expressed in leaf and flower abscission zones and in pistils of fully open flowers. TAPG4 mRNA is detected much earlier than TAPG1 and TAPG2 mRNA during both leaf and flower abscission.     

Abscission of Azolla branches induced by ethylene and sodium azide

Treatment with ethylene accelerated the abscission of branches of Azolla filiculoides plants. An Azolla plantlet treated with ethylene at 10 microl liter(-1) divided into 4-5 fragments after a lag period of 6-8 h. Ethylene-induced abscission was effectively inhibited by cycloheximide and was associated with an increase in the activities of cellulase and polygalacturonase. At the fracture surface abscised after treatment with ethylene, dissolution of the primary walls of the abscission zone cells was apparent. However, the middle lamella between abscission zone cells was still present. Immunoelectron microscopy using anti-unesterified pectin (JIM5) and anti-methylesterified pectin (JIM7) monoclonal antibodies revealed the presence of both JIM5 and JIM7 epitopes in the wall between abscission zone cells of branches before abscission occurred. In the middle lamella remaining after ethylene-induced abscission, only JIM7 epitopes were observed. The features of ethylene-induced abscission described herein were different from those of the rapid abscission induced by sodium azide, which implies that they are mediated by different mechanisms. The possible mechanisms are discussed.