Leaf Abscission Zones Research Articles

Characterization of abscission zones in the flowers and fruits of peach [Prunus persica (L.) Batsch

Cell wall hydrolases, their mRNAs, and ultrastructural details of cell wall digestion have been studied in peach abscission zones (AZ) located at the base of flower bud (AZ1) and the base of flower receptacle (AZ2), respectively. Induction of abscission was obtained by treatment of explants with exogenous ethylene. Cell separation patterns of the two examined abscission zones have been compared with those of other already known AZs of peach, i.e. the AZs located between fruit and peduncle and the leaf AZ. Analyses have shown similarities in response to ethylene treatment between AZ1 and leaf AZ and between AZ2 and AZs, respectively. Results have been discussed considering the precise position of AZ1 and AZ2 on the flower bud. The timing of functional differentiation, evaluated as the cells'ability to respond to induction by ethylene treatments, showed that AZ1 and AZ2 became functional after bud breaking and bud scale shedding. Later on, they lost their functionality at about 6-7 wk from anthesis. AZ3 became functional very precociously and could be activated 1 wk after anthesis in the fertilized flowers. In the latter zone the cells could also undergo a morphological predifferentiation, even though it occurred a long time after the acquisition of the ethylene responsiveness. This finding shows that morphological differentiation is not necessarily a prerequisite for those cells to become competent to respond to the abscission inducing stimuli.

Physiology of Olive Leaf Abscission Induced by Phosphorus

Adding Al2O3 to 8-hydroxyquinoline citrate (8-HQC) solution did not alter the sensitivity of the leaf abscission zone to external ethylene. Exogenous ethylene at 791 nl·liter-1 for 72 to 120 hours and at 193 nl·liter-1 for 120 hours induced leaf abscission, whereas no leaf abscission occurred at 47 nl·liter-1 for 72 to 120 hours. Ethylene at 791 nl·liter-1 for 72 to 120 hours increased ethylene evolution, but the amount of ethylene evolved from the explants does not seem to be enough to induce leaf abscission. Three different ethylene inhibitors—aminooxyacetic acid (AOA), CoCl2, and aminoethoxyvinylglycine (AVG)—were used to determine whether P-induced leaf abscission was mediated through elevated ethylene evolution. Although AOA and CoCl2 failed to inhibit ethylene evolution from the explants stem-fed with NaH2PO4, AVG inhibited ethylene evolution. Each inhibitor, except 5 mm CoCl2, promoted leaf abscission when administered alone or with P. Our results reveal that P-induced olive leaf abscission may occur without elevated ethylene evolution. At 40 or 75 mm NaH2PO4, abscission did not occur until explants were removed from N2 and placed in ambient air.

754 PB 146 Physiology of Olive Leaf Abscission Induced by Phosphorus

The addition of Al2O3 to 8-hydroxyquinoline citrate (8-HQC) solution did not alter the sensitivity of the leaf abscission zone to external ethylene treatment. Exogenous ethylene at 791 nl·l-1 for 72 to 120h and at 193 nl·l-1 for 120h induced leaf abscission whereas at 47 nl·l-1 for 72 to 120h no leaf abscission occurred. Ethylene treatment at 791 nl·l-1 for 72 to 120h increased ethylene evolution, but the amount of ethylene evolved from the explants does not seem to be enough for leaf abscission induction. Three different ethylene inhibitors, aminooxyacetic acid (AOA), CoCl2 and am inoethoxyvinylglycine (AVG), were used to determine whether phosphorus-induced leaf abscission was mediated through elevated ethylene evolution. Although AOA and CoCl2 failed to inhibit ethylene evolution from the explants stem-fed with NaH2P O4, AVG inhibited ethylene evolution. Each of the inhibitors except for 5 mM CoCl2 promoted leaf abscission when administered alone or with phosphorus. Our results reveal that phosphorus induced olive leaf abscission occurs without elevated ethylene evolution, but that oxygen is required.

Characterization and accumulation pattern of an mRNA encoding an abscission-related ?-1,4-glucanase from leaflets of Sambucus nigra

A cDNA library was produced using mRNA extracted from ethylene-treated leaflet abscission zones of common elder (Sambucus nigra). Screening of the library with the insert from pBAC10, which encodes an abscission beta-1,4-glucanase (cellulase) from bean (Phaseolus vulgaris), resulted in the isolation of a near-full-length cDNA which was designated JET 1. Northern analysis, using JET 1 as a probe, detected a transcript of 1.9 kb that accumulated prior to the first visible signs of cell separation. Accumulation of the JET 1 transcript is promoted by ethylene and primarily restricted to the tissue comprising the abscission zone. Sequence analysis of JET 1 indicates it is 1768 bp in length and shares significant homology at the amino acid level with beta-1,4-glucanases from the leaf abscission zone of P. vulgaris (67%) and ripening avocado fruit (48%). The predicted peptide sequence of the S. nigra enzyme contains two potential glycosylation sites. Genomic Southern analysis of S. nigra DNA reveals that JET 1 may belong to a multi-gene family.

Cellulase and polygalacturonase involvement in the abscission of leaf and fruit explants of peach

Ethylene-induced abscission in leaf and fruit explants of peach involves different enzymes. In leaves abscission is accompanied by increased occurrence of cellulase forms differing in isoelectric point (pI 6.5 and 9.5). A polypeptide with a molecular mass of 51 kDa gives in a western blot a strong cross-reaction with an antibody raised against a maturation cellulase from avocado fruit. Cellulase activity is also found in abscising fruit explants but the amount is very low compared to that of the leaf explants. A northern analysis with a cellulase clone from avocado reveals the presence of two hybridizing mRNAs with a size of 2.2 kb and 1.8 kb, respectively. The steady-state level of the 2.2 kb mRNA is significantly increased by treatment with ethylene. Polygalacturonases are not detected in abscising leaves, but are strongly induced by ethylene in fruit explants. Of the three forms found, two are exopolygalacturonases while the third is an endoenzyme. Ethylene activates preferentially the endoenzyme and the basic exoenzyme but depresses the acid exopolygalacturonases. A northern analysis carried out with a cDNA coding for tomato endopolygalacturonase shows hybridization only with one endopolygalacturonase mRNA form in the fruit abscission zone. Treatment with ethylene causes an increase in the steady-state level of this mRNA. The differences in the enzyme patterns observed in fruit and leaf abscission zones and a differential enzyme induction suggest the feasibility to regulate fruit abscission in peach with the aid of antisense RNA genes.

Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants

Polygalacturonase (PG, EC 3.2.1.15), an enzyme commonly found in ripening fruit, has also been shown to be associated with abscission. A zone-specific rise in PG activity accompanies the abscission of both leaves and flowers of tomato (Lycopersicon esculentum Mill.) plants. Studies of transgenic plants expressing an antisense RNA for fruit PG indicate that although the enzyme activity in transgenic fruit is < 1 % of that in untransformed fruit, the PG activity in the leaf abscission zone increases during separation to a similar value to that in untransformed plants. The timing and rate of leaf abscission in transgenic plants are unaffected by the introduction of the antisense gene. A polyclonal antibody raised against tomato fruit PG does not recognise the leaf abscission protein. Furthermore a complementary DNA (cDNA) clone (pTOM6), which has been demonstrated to code for fruit PG, does not hybridise to mRNA isolated from the abscission-zone region of tomato leaves. These results indicate that the PG protein in abscission zones of tomato is different from that in the fruit, and that the gene coding for this protein may also be different.

Identification of chitinase mRNA in abscission zones from bean (Phaseolus vulgaris Red Kidney) during ethylene‐induced abscission

Abstract. Total RNA was extracted from bean leaf abscission zones at different times after the induction of abscission by ethylene. The RNA was translated in the wheat germ system and the products analysed by SDS‐PAGE. Products of molecular weight (raw) 42, 32 and 17 kD were seen to accumulate substantially during the induction. An attempt was made to establish that the mRNA species which produced the 32 kD product, which was coded for the ethylene‐regulated enzyme chitinase. Mature chitinase (30 kD) was purifed from ethylene‐treated abscission zones and used to raise monospecific antibodies in rabbits. These antibodies recognized the 32 kD product and mature chitinase. The 2 kD difference in molecular weight was due to the presence of the signal sequence which could be removed by microsomal membranes. Chitinase was also detected by enzymatic assay and immunoblotting of crude homogenates from ethylene‐treated abscission zones. Chitinase appears to be ubiquitous in bean plants and probably does not have a direct role in abscission.

Positional control of differentiation of ethylene responsive target cells in leaf abscission zones: [formula omitted]1 and [formula omitted]2. 1Developmental Botany, AFRC Weed Research Organization, Oxford OX5 1PF, UK. 2Biochemistry Department, University of Oxford, Oxford OX1 3QU, UK

Positional control of differentiation of ethylene responsive target cells in leaf abscission zones: [formula omitted]1 and [formula omitted]2. 1Developmental Botany, AFRC Weed Research Organization, Oxford OX5 1PF, UK. 2Biochemistry Department, University of Oxford, Oxford OX1 3QU, UK

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.

Some histochemical observations on leaf abscission zone inCapsicum annuum L.

The present observations were conducted on NP 46-A and Pusa jwala varieties ofCapsicum annuum L. Investigations on changes in cell wall components of the cells of leaf abscission zone associated with the process of abscission were based on specific qualitative histochemical staining procedures. It was observed that the loss of pectic and cellulosic substances preceded the separation phase of abscission from middle lamella and cell walls of the cells of separation layer of abscission zone. The abscission zone was characterized by thin cutin deposition on epidermal cells and poor lignification in vascular elements of this region. Separation of senescent part of petiole was followed by deposition of suberin in cells of some outer layers of abscission zone and by the deposition of lignin in cells of the remaining layers of abscission zone to form a protection layer on exposed part of petiole stump. Development of tyloses, tannins and calcium oxalate crystals was not found associated with abscission in the present plant material.

Localized cell growth in ethephon-treated olive leaf abscission zone

Ethephon-induced growth of olive-leaf abscission-zone cells is limited to the cortical separation layer and cells surrounding indentations present at the abaxial and adaxial surfaces of the petiole base. Proximal and distal abscission-zone cells on either side of the separation layer do not enlarge. Enlargement of cortical cells and of cells surrounding the abaxial notch begins by 24 h after ethephon treatment. Cell enlargement in the region of the adaxial indentation, the final region to undergo separation, does not begin until after the first 24 h. Prior to separation, separation-layer cell walls swell to more than 3 times their original thickness, and cell wall thickness decreases following separation. Nuclei of separation-layer cells are smaller than those of adjacent abscission-zone cells.

Role of Polygalacturonase in Bean Leaf Abscission

The role of polygalacturonase in leaf abscission was studied in explants of Phaseolus vulgaris L. cv. Red Kidney. Bean polygalacturonase was partially characterized and comparisons were made between the bean enzyme and previously reported higher plant polygalacturonases. Polygalacturonase isolated from bean leaf abscission zones has a pH optimum between 4.5 and 5.0 and hydrolyzed polygalacturonides in an exo-fashion. Activity was found to be higher with a deesterified substrate than with an esterified pectin. No correlation between polygalacturonase activity and abscission was observed. Activity remained virtually constant over the course of abscission in explants aged either in air or in ethylene. The enzyme was primarily localized in the abscission zone, however, indicating a possible involvement in the abscission process. A theoretical model which could explain the relationship between polygalacturonase and bean leaf abscission is discussed.

ULTRASTRUCTURAL STUDIES OF ABSCISSION IN PHASEOLUS: LOCALIZATION OF PEROXIDASE

Segments of leaf abscission zone tissue of Phaseolus vulgaris L. cv. Red Kidney were fixed in glutaraldehyde, incubated to demonstrate peroxidase activity in medium containing 3,3'‐diaminobenzidine (DAB) and postfixed in osmium tetroxide. Electron microscopic observation of treated tissue revealed pronounced deposition of highly electron‐opaque material in the form of granules or globules in cell walls, on mitochondrial membranes, associated with rough endoplasmic reticulum and along the plasmalemma and tonoplast. This distribution pattern was typical of both non‐treated and ethephon‐treated tissue. Ethephon‐treated material also contained these granules within cytoplasmic vacuoles. It is suggested that pH of the incubation medium may affect localization sites and that exposure of tissue to light during incubation may modify localization patterns. Differing patterns of distribution of the reaction product in treated and non‐treated tissue may reflect changes in membrane permeability and microfibrillar modifications related to ethephon treatment.

Cell Separation in Isolated Abscission Zones

A technique is described for the isolation and maintenance culture of 250 p;m thick slices of the leaf abscission zone of Ooleus. Such cultures can be observed under the light microscope and a continuous photographic record of the cytological events associated with abscission can be obtained. Certain stages of the process can be accurately defined and the opportunity arises for closely correlated cytological and physiological studies by exposing the tissues to agents known to influence abscission. Some examples illustrating these points are discussed briefly