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.     

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 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.     

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.     

Occurrence and Localization of 9.5 Cellulase in Abscising and Nonabscising Tissues

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.     

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.     

Jasmonates promote abscission in bean petiole expiants: Its relationship to the metabolism of cell wall polysaccharides and cellulase activity

Jasmonic acid (JA) and its methyl ester (JA-Me) promoted the abscission of bean petiole expiants in the dark and light, and the activity of these compounds was almost same. JA and JA-Me did not enhance ethylene production in bean petiole expiants in the light, indicating that the abscission-promoting effects of these compounds are not the result of ethylene. Cells in the petiole adjacent to the abscission zone expanded during abscission but not in the pulvinus, and JA-Me promoted cell expansion in the petiole and the pulvinus. JA-Me had no effect on the total amounts of pectic and hemicellulosic polysaccharides in 2-mm segments of the abscission region, which included 1 mm of pulvinus and 1 mm of petiole from the abscission zone. On the other hand, the total amounts of cellulosic polysaccharides in this region were reduced significantly by the addition of JA-Me in the light. JA-Me had no effect on the neutral sugar composition of hemicellulosic polysaccharides during abscission. The decrease in the endogenous levels of UDP-sugars in the petiole adjacent to the abscission zone was accelerated during abscission by the addition of JA-Me in the light. Cellulase activities of pulvinus and petiole in 10-day-old seedlings were enhanced by the addition of JA. These results suggest that the promoting effect of JA or JA-Me on the abscission of bean petiole explants is due to the change of sugar metabolism in the abscission zone, in which the increase in cellulase activity involves the degradation of cell wall polysaccharides. Jasmonic acid (JA) and its methyl ester (JA-Me) are considered to be putative plant hormones for a number of reasons, including their wide occurrence in the plant kingdom, biologic, activities in multiple aspects at low concentrations, and their interaction with other plant hormones (for reviews see Parthier 1991, Hamberg and Gardner 1992, Sembdner and Parthier 1993, Ueda et al. 1994a). We have already reported that JA and JA-Me and C₁₈-unsaturated fatty acids, which are considered to be the substrates of the biosynthesis of jasmonates, are powerful senescence-promoting substances (Ueda et al. 1982b, 1991a). Senescence symptoms induced by these compounds are identical to those of natural senescence. Recently we have also found that JA inhibited indole-3-acetic acid (IAA)-induced elongation of oat (Avena sativa L. cv. Victory) coleoptile segments by inhibiting the synthesis of cell wall polysaccharides (Ueda et al. 1994b, 1995). These facts led us to study the mode of actions of JA and JA-Me on promoting abscission, which is considered the last dramatic phenomenon of senescence. In this paper we report that JA and JA-Me promote abscission in bean (Phaseolus vulgaris L. cv. Masterpiece) petiole expiants and that the changes in the metabolism of cell wall polysaccharides in the petiole and the pulvinus adjacent to the abscission zone are involved in the promotive effects of these compounds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - DCB 2,6-dichlorobenzonitrile - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - JA jasmonic acid - JA-Me methyl jasmonate - MES 2-(N-morpholino)ethane-sulfonic acid, monohydrate - TCA trichloroacetic acid - Tris 2-amino-2-hydroxymethy-1,3-propanediole     

Failed expression of an endo-beta-1,4-glucanhydrolase (cellulase) in a non-abscinding mutant of Lupinus angustifolius cv Danja.

Cellulase expressions in a normal shedding wild-type and a non-abscinding single gene mutant of Lupinus angustifolius have been studied during ethylene treatments of leaf abscission zone explants. Of the range of different glycohydrolases investigated only the abscission cell-specific beta-1,4-glucanhydrolase (cellulase) was not produced in the non-abscinding mutant. An endo-polygalacturonase was induced equally in both wild-type and mutant and other glycohydrolases were equally up-regulated. The abscission cell-specific cellulase induced at shedding of wild-type is antigenically similar to the Phaseolus vulgaris induced leaf abscission pI 9.5 cellulase but with a higher molecular mass (50 kD compared with 48 kD) and like the bean abscission-specific cellulase that of lupin is not glycosylated. Causes of the loss of function of cellulase expression in the non-shedding mutant are 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.     

A Xyloglucan-Specific Endo-1,4-[beta]-Glucanase Isolated from Auxin-Treated Pea Stems

A xyloglucan-specific endo-1,4-[beta]-glucanase was isolated from the apoplast fraction of auxin-treated pea (Pisum sativum) stems, in which both the rate of stem elongation and the amount of xyloglucan solubilized were high. The enzyme was purified to apparent homogeneity by sequential cation-exchange chromatographies, affinity chromatography, and gel filtration. The purified enzyme gave a single protein band on sodium dodecyi sulfate-polyacrylamide gel electrophoresis, and the molecular size was determined to be 77 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 70 kD by gel filtration. The isoelectric point was about 8.1. The enzyme specifically cleaved the 1,4-[beta]-glucosyl linkages of the xyloglucan backbone to yield mainly nona- and heptasaccharides but did not hydrolyze carboxymethylcellulose, swollen cellulose, and (1->3, 1->4)-[beta]-glucan. By hydrolysis, the average molecular size of xyloglucan was decreased from 50 to 20 kD with new reducing chain ends in the lower molecular size fractions. This suggests that the enzyme has endo-1,4-[beta]-glucanase activity against xyloglucan. In conclusion, a xyloglucan-specific endo-1,4-[beta]-glucanase with an activity that differs from the activities of cellulase and xyloglucan endotransglycosylase has been isolated from elongating pea stems.     

Abscission: the role of aging

Excision of Phaseolus vulgaris L. c.v. Red Kidney abscission zone explants results in senescence, mobilization, and abscission. Because these processes take place at about the same time, there has been some question as to whether they are causally related or are occurring in an independent but simultaneous fashion. Data presented here suggest that the latter interpretation is correct. After abscission zone explants are isolated from the leaf an aging process is set into motion and a degradation of metabolites in the pulvinus takes place. During the aging process the explants also become increasingly sensitive to ethylene which in turn promotes cell separation. Indoleacetic acid, cytokinins, and coumarin appear to retard aging since both degradative processes and abscission are inhibited. However, ethylene increased abscission without increasing degradative processes indicating that abscission and senescence are independent processes occurring at the same time.     

Cellulase, Fruit Softening and Abscission in Red RaspberryRubus idaeusL. cv Glen Clova

The ripening of raspberry fruit (Rubus ideausL. cv Glen Clova)is associated with a climacteric rise in ethylene production.As the fruit pigments change from green to red there is a progressivesoftening, loss of skin strength and a breakdown of cell wallsin the mesocarp. An increase in cellulase (endo-1,4-ß-D-glucanase)in both drupelets and receptacles accompanies these changes.The localization of cellulase in the regions of the fruit associatedwith abscission zones suggest the enzyme may be involved infruit separation as well as softening. Rubus idaeusL; raspberry; fruit ripening; ethylene; abscission; cell wall breakdown; cellulase; endo-1,4-ß-D-glucanase     

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.     

Purification of β1,4 glucanase from ethylene-treated leaflet abscission zones of Sambucus nigra

Leaflet abscission in Sambucus nigra is the result of cell wall breakdown at the site of separation. Associated with wall degradation is an increase in the activity of the enzyme β1,4 glucanase (E.C.3.1.2.4) in the cells that comprise the abscission zone. The enzyme has been extracted from abscission zone tissue and purified using a substrate affinity column. A qualitative enzyme assay procedure has been developed and this has facilitated the purification process. The β1,4 glucanase enzyme has a pH optimum of 7 and a molecular mass of 54kDa. Antibodies have been raised to the purified protein. The role of the enzyme in the abscission process is discussed.     

Degradation of the upper pulvinus in modern and fossil leaves of Cercis (Fabaceae)

Identification of fossil leaf impressions as Cercis has been questioned based upon the presence or absence of a pulvinus at the base of the lamina (upper pulvinus). In the present study, leaves of Cercis canadensis were examined before and after abscission to explore the degradation processes that could occur prior to fossilization, and the North American record for fossil foliage of Cercis was revised accordingly. Results for C. canadensis indicate that: (1) the pulvinus consists largely of tissues with nonlignified cells (a wide cortex, a nonlignified fiber sheath, phloem, and pith) that degrade rapidly after leaf abscission, (2) the lignified xylem tissue that remains in the pulvinus after degradation is in brittle strands, (3) the pulvinus degrades at a faster rate than the lamina or the petiole, and (4) the degraded pulvinus cushion leaves a semicircular pattern on the lamina. From examination of fossils as well as extant species, we: (1) demonstrated that in fossils, the upper pulvinus can show a greater degree of degradation than the adjoining petiole or lamina tissue, suggesting the degradation of upper pulvinus tissue is similar in modern vs. fossil specimens, (2) defined numerous other laminar characters that can be used in conjunction with, or in the absence of, an upper pulvinus to confirm the presence of Cercis in the fossil record, and (3) showed from those criteria that the earliest known North American fossil leaf record for Cercis, from a specimen newly reported in the present study, is from the middle Miocene Succor Creek flora of Oregon.     

Separation of vascular cell walls from cortical cell walls of plant roots

Summary The cortex was physically separated from the stele of corn roots. The isolated walls from cortical cells were less dense than the walls isolated from stelar cells. The cell walls from each tissue were centrifuged on a step gradient composed of 50 and 60% sucrose for 5 min at 900 g. After the short centrifugation time, the cortical cell walls banded at the 50/60% interface while the vascular tissue walls pelleted through 60% sucrose. An aliquot of vascular cell walls was then marked with cytochromec. The marked cell walls were mixed with cortical cell walls and centrifuged on a 50/60% sucrose gradient and after 5 min, the vascular tissue walls were cleanly separated from the cortical cell walls. The experiment was repeated without prior separation of tissue types with another corn variety, carrot roots grown in culture, and pea roots. A clean separation of cell wall types was achieved after homogenization of intact roots in liquid nitrogen, extrusion from a nitrogen bomb, and centrifugation in sucrose gradients.     

Changes in Polygalacturonase Activity and Solubility of Polyuronides during Ethylene-stimulated Leaf Abscission in Sambucus nigra

Leaflet abscission in Sambucus nigra is precipitated by cellwall degradation which is restricted to the site of cell separation.Accompanying wall breakdown is an increase in the activity ofthe enzyme polygalacturonase (PG) (E.C. 3.2.1.15 [EC] ) and this riseis primarily confined to the abscission zone tissue. The polygalacturonasehas a pH optimum of 4·4 and has the characteristics ofan endo-acting enzyme. The elevation in enzyme activity is theresult of an increase in at least two isoforms of PG as revealedby polyacrylamide gel electrophoresis of the natured protein.Leaflet abscission in S. nigra is associated with an increasein the solubility and depolymerization of polyuronides fromthe cell wall. These observations are discussed in relationto the mechanism of cell separation during ethylene-stimulatedleaf abscission. Key words: Elder, Sambucus nigra, abscission, polygalacturonase, polyuronides, ethylene     

Spatial and temporal aspects of cell separation in the foliar abscission zones ofImpatiens sultani Hook

Summary The abscission of leaves fromImpatiens sultani Hook. occurs as the direct result of the weakening of a narrow band of cells running transversely across the base of the petiole. This loss of strength of the abscission zone is due to the breakdown of the central cell wall in two or three layers of cells. The process of wall degeneration is first visible 13 hours after the induction of abscission in a small group of cells found just below the concave groove on the adaxial side of the petiole. As the abscission zone gets progressively weaker the area of cells showing wall breakdown expands, spreading through the parenchyma to the lower side of the stele. The walls of the collenchyma and epidermis along the sides and base of the petiole and the central vascular tissues are the last to break down. Experiments in which the abscission zone was dissected into small pieces were undertaken to investigate whether cell wall hydrolysis was a contagious phenomenon, spreading from cell to cell by direct contact. The results of these investigations indicated that there was little requirement for cell to cell contact in either the temporal or spatial integration of cell wall breakdown.     

Antisense suppression of tomato endo-1,4-β-glucanase Cel2 mRNA accumulation increases the force required to break fruit abscission zones but does not affect fruit softening

Plants of tomato (Lycopersicon esculentum Mill. cv. T5) were transformed with an antisense endo-1,4--glucanase (cellulase, EC 3.2.1.4) Cel2 transgene under the control of the constitutive cauliflower mosaic virus 35S promoter in order to suppress mRNA accumulation of Cel2. In two independent transgenic lines, Cel2 mRNA abundance was reduced by >95% in ripe fruit pericarp and ca. 80% in fruit abscission zones relative to non-transgenic controls. In both transgenic lines the softening of antisense Cel2 fruit pericarp measured using stress-relaxation analysis was indistinguishable from control fruit. No differences in ethylene evolution were observed between fruit of control and antisense Cel2 genotypes. However, in fruit abscission zones the suppression of Cel2 mRNA accumulation caused a significant (P<0.001) increase in the force required to cause breakage of the abscission zone at 4 days post breaker, an increase of 27% in one transgenic line and of 46% in the other transgenic line. Thus the Cel2 gene product contributes to cell wall disassembly occurring in cell separation during fruit abscission, but its role, if any, in softening or textural changes occurring in fruit pericarp during ripening was not revealed by suppression of Cel2 gene expression.