A polygalacturonase from citrus leaf explants: role in abscission

The relationship between polygalacturonase activity and abscission of citrus leaf explants was studied. Determination of polygalacturonase activity in citrus tissues requires concentration of the enzyme, use of a proper assay method, and inhibition of an oxidase present in the extracts which oxidizes the reaction products of the polygalacturonase. The polygalacturonase from citrus leaf explants is an exopolygalacturonase and appears to be a soluble enzyme.     

Abscission of Citrus Leaf Explants: INTERRELATIONSHIPS OF ABSCISIC ACID, ETHYLENE, AND HYDROLYTIC ENZYMES

The question whether abscisic acid (ABA) induces cellulase and polygalacturonase activity and, hence, abscission directly or whether its action is mediated by C₂H₄ was studied in citrus (Osbeck var. Shamouti) leaf explants using aminoethoxyvinyl glycine (AVG), an inhibitor of C₂H₄ biosynthesis. ABA in concentrations of 10 micromolar and higher induced C₂H₄ production and accelerated abscission. AVG inhibited C₂H₄ formation, activity of cellulase and polygalacturonase, and abscission in ABA-treated explants. AVG did not inhibit the increase in the activity of the cell-wall degrading enzymes or abscission in a saturating level of externally supplied C₂H₄. This indicates that the effect of AVG resulted from inhibition of the formation of endogenous ethylene. The data indicate that in citrus leaf explants the induction of the activity of cellulase and polygalacturonase and abscission by ABA is mediated by C₂H₄.     

Enhancement of RNA synthesis, protein synthesis, and abscission by ethylene

Ethylene stimulated RNA and protein synthesis in bean (Phaseolus vulgaris L. var. Red Kidney) abscission zone explants prior to abscission. The effect of ethylene on RNA synthesis and abscission was blocked by actinomycin D. Carbon dioxide, which inhibits the effect of ethylene on abscission, also inhibited the influence of ethylene on protein synthesis. An aging period appears to be essential before bean explants respond to ethylene. Stimulation of protein synthesis by ethylene occurred only in receptive or senescent explants. Treatment of juvenile explants with ethylene, which has no effect on abscission also has no effect on protein synthesis. Evidence in favor of a hormonal role for ethylene during abscission is discussed.     

Bean abscission cellulase : characterization of a cDNA clone and regulation of gene expression by ethylene and auxin

The physiology and anatomy of abscission has been studied in considerable detail; however, information on the regulation of gene expression in abscission has been limited because of a lack of probes for specific genes. We have identified and sequenced a 595 nucleotide bean (Phaseolus vulgaris cv Red Kidney) abscission cellulase cDNA clone (pBACl). The bean cellulase cDNA has extensive nucleic and amino acid sequence identity with the avocado cellulase cDNA pAV363. The 2.0 kilobase bean mRNA complementary to pBACl codes for a polypeptide of approximately 51 kilodalton (shown by hybrid-selection followed by in vitro translation). Bean cellulase antiserum is shown to immunoprecipitate a 51 kilodalton polypeptide from the in vitro translation products of abscission zone poly(A)⁺ RNA. Ethylene initiates bean leaf abscission and tissue-specific expression of cellulase mRNA. If ethylene treatment of bean explants was discontinued after 31 h and then 2,5-norbornadiene given to inhibit responses resulting from endogenously synthesized ethylene, polysomal cellulase mRNA hybridizing to pBACl decreased. Thus, ethylene is required not only to initiate abscission and cellulase gene expression but also to maintain continued accumulation of cellulase mRNA. Explants treated with auxin 4 hours prior to a 48 hour treatment with ethylene showed no substantial accumulation of RNA hybridizing to pBACl or expression of cellulase activity.     

Abscission of citrus leaf explants: no correlation with naphthaleneacetic Acid conjugation in the abscission zone

The role of α-naphthaleneacetic acid (NAA) in the control of abscission in Citrus (Citrus sinensis L. Osbeck) leaf explants and its conjugation were studied in non-aged and 24-hour-aged explants. Dipping non-aged explants in 1.5 micromolar NAA for 15 minutes immediately after excision did not delay abscission whereas 150 micromolar NAA effectively delayed it. As incubation time was prolonged up to 24 hours after excision, the delaying effect of both concentrations gradually increased. In general, both concentrations did not delay abscission when applied to 24-hour-aged explants held for an additional period of up to 24 hours. The uptake and conjugation of ¹⁴C-NAA to glucose and aspartic acid were similar in petiole, abscission zone, and leaf blade of non-aged and aged tissues, for all NAA concentrations. No correlation was established between the kinetics of abscission and the rate of conjugation in the abscission zone.     

Optimal and Spatial Analysis of Hormones,Degrading Enzymes and Isozyme Profiles in Tomato Pedicel Explants During Ethylene-Induced Abscission

Activities of degrading enzymes, hormones concentration and zymogram patterns were investigated during control and ethylene-induced abscission of tomato pedicel explants. Exogenous ethylene accelerated abscission of pedicel explants. It was showed that IAA concentration in abscission zone tended to decline at first and then was reduced before separation in control and ethylene-treatment. Moreover, IAA (indole acetic acid) and ABA (abscise acid) concentrations were elevated in each segment when exposing to ethylene, but GA_{1 + 3} (gibberellin1 + gibberellin3) concentration was decreased in abscission zone and the proximal side. Activities of cellulase, polygalacturonase and pectinesterase in the explants were induced in the separating process and strengthened by ethylene. However, comparing with the proximal side, cellulase and polygalacturonase activities in abscission zone and distal side were higher. Electrophoresis of isozymes revealed that at least three peroxidase and three superoxidase isozymes appeared in the explants, respectively. One peroxidase isozyme exhibited differentially among the three positions in control and ethylene-treatment. One esterase isozyme weakened or disappeared in the following hours, but three novel esterase isozymes were detectable from beginning of the process. The data presented support the hypothesis that the distal side, together with abscission zone of explants plays a more important role in separation than does the proximal side. The possible roles of degrading enzymes, hormones and isozymes in three segments during ethylene-induced abscission of tomato pedicel explants are discussed.     

Flower abscission in mutant tomato plants

The effect of two mutations of the tomato known as Never ripe (Nr) and ripening inhibitor (rin) on abscission of the flowers was investigated. In the presence of ethylene the rate of abscission of normal and rin explants was similar, while that of Nr explants was delayed. The appearance and subsequent increases in both polygalacturonase (EC 3.2.1.15) and -1-4-glucanase (EC 3.2.1.4) enzyme activities were similar in normal and rin explants, but retarded in Nr explants. Of these two, only polygalacturonase activity was exclusively associated with abscission-zone tissue.Abbreviations PG Polygalacturonase - Nr Never ripe mutation - rin ripening inhibitor mutation     

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.     

Ethylene-induced leaf abscission in cotton seedlings : the physiological bases for age-dependent differences in sensitivity

The speed of ethylene-induced leaf abscission in cotton (Gossypium hirsutum L. cv LG-102) seedlings is dependent on leaf position (i.e. physiological age). Fumigation of intact seedlings for 18 hours with 10 microliters per liter of ethylene resulted in 40% abscission of the still-expanding third true (3°) leaves but had no effect on the fully expanded first true (1°) leaves. After 42 hours of fumigation with 50 microliters per liter of ethylene, total abscission of the 3° leaves occurred while <50% abscission of the 1° leaves was observed. On a leaf basis, endogenous levels of free IAA in 1° leaves were approximately twice those of 3° leaves. Free IAA levels were reduced equally (approximately 55%) in both leaf types after 18 hours of ethylene (10 microliters per liter) treatment. Ethylene treatment of intact seedlings inhibited the basipetal movement of [¹⁴C]IAA in petiole segments isolated from both leaf types in a dose-dependent manner. The auxin transport inhibitor N-1-naphthylphthalamic acid increased the rate and extent of ethylene-induced leaf abscission at both leaf positions but did not alter the relative pattern of abscission. Abscission-zone explants prepared from 3° leaves abscised faster than 1° leaf explants when exposed to ethylene. Ethyleneinduced abscission of 3° explants was not appreciably inhibited by exogenous IAA while 1° explants exhibited a pronounced and protracted inhibition. The synthetic auxins 2,4-D and 1-naphthaleneacetic acid completely inhibited ethylene-induced abscission of both 1° and 3° explants for 40 hours. It is proposed that the differential abscission response of cotton seedling leaves is primarily a result of the limited abscission-inhibiting effects of IAA in the abscission zone of the younger leaves.     

Deferral of senescence and abscission by chemical inhibition of ethylene synthesis and action in bean explants

Three compounds known to inhibit ethylene synthesis and/or action were compared for their ability to delay senescence and abscission of bean explants (Phaseolus vulgaris L. cv Contender). Aminoethoxyvinyl-glycine (AVG), AgNO₃, and sodium benzoate were infiltrated into the petiole explants. Their effect on abscission was monitored by measuring the force required to break the abscission zone, and their effect on senescence was followed by measuring chlorophyll and soluble protein in the distal (pulvinus) sections. AVG at concentrations between 1 and 100 micromolar inhibited ethylene synthesis by about 80 to 90% compared to the control during sampling periods of 24 and 48 hours after treatment. This compound also delayed the development of abscission and senescence. Treatment with AgNO₃ at concentrations between 1 and 100 micromolar progressively reduced ethylene production, but to a lesser extent than AVG. The effects of AgNO₃ on senescence and abscission were quite similar to those of AVG. Sodium benzoate at 50 micromolar to 5 millimolar did not inhibit ethylene synthesis during the first 24 hours, but appreciably inhibited ethylene synthesis 48 hours after treatment. It also delayed the development of abscission and senescence. The effects of AVG, Ag⁺, and sodium benzoate suggest that ethylene could play a major role in both the senescence induction phase and the separation phase in bean explants.     

Abscission: characterization of light control

Exposure of mung bean (Vigna radiata [L.] Wilczek cv Jumbo) cuttings to low level red light inhibits dark-induced leaf abscission. A 12-hour daily light requirement for maximum inhibition of abscission was equally effective as a continuous red light treatment or shorter light-dark cycles. Transfer of cuttings from dark to light stopped the abscission process at the time of transfer. The available evidence suggests a light receptor located in the leaves with an abscission inhibitor translocated from lighted leaf to the abscission zone.     

Abscission: movement and conjugation of auxin

A 1-hour application of indole-3-acetic acid to bean (Phaseolus vulgaris L. cv. Red Kidney) explants inhibited abscission for an 8-hour aging period. Use of indole-3-acetic acid-¹⁴C showed that the applied indole-3-acetic acid was conjugated within explant tissue and that this conjugation mechanism accounts for loss of effectiveness of indole-3-acetic acid in inhibiting abscission after 8 hours. Reapplication of indole-3-acetic acid to an explant at a later time, before the induced aging requirement was completed reinhibited abscission. 2,4-Dichlorophenoxyacetic acid, which is not destroyed or conjugated by this system, did not lose its ability to inhibit abscission. It was concluded that indole-3-acetic acid destruction is one of the processes involved in the aging stage of abscission in explants.     

Pectin esterase in relation to leaf abscission in coleus and phaseolus

Pectin esterase (PE) activities in abscission zones, other portions of leaves, and adjacent stem tissues were compared in attached leaves and abscissing petioles (previously debladed) of Coleus blumei Benth. and Phaseolus vulgaris L., cv. Canadian Wonder. Earlier findings of Osborne in bean were confirmed and changes in PE activity in coleus were shown to resemble those in bean in some respects. In both plants PE was lower in the distal portion of abscission zones of abscissing petioles than in that portion of attached leaves but this difference was not as large or as consistently clear-cut in coleus as in bean. The general level of PE activity was an order of magnitude lower and changes associated with abscission were smaller in coleus than in bean. Auxin treatment of debladed petioles of coleus prevented abscission and resulted in small increases in PE activity in abscission zones and most of the other regions sampled. The largest increase was observed in the stem tissue adjacent to the attached leaf opposite the debladed, auxin treated one.     

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 influence of auxin, cacodylic Acid, and amitrole on the abscission of petiole explants

The influence of indoleacetic acid, cacodylic acid (hydroxy-dimethylarsine oxide), and amitrole (3-amino-1,2,4-triazole) on the petiole explant abscission rate was studied in three species. Indoleacetic acid increased the abscission rate in both bean (Phaseolus vulgaris L. var. Red Kidney) and Coleus (Coleus blumei Benth) at 10⁻³ and 10⁻⁴m but had no effect on abscission in privet (Ligustrum ovalifolium). Cacodylic acid was found to stimulate abscission in explants of beans and privet, but not in Coleus. Amitrole did not stimulate abscission under any circumstance tested. In no case was the abscission rate dependent on the time at which any of the chemicals was applied. These data do not support the two-phase response of explants to applied auxin.     

Involvement of ethylene in the action of the cotton defoliant thidiazuron

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