No abstract

The tomato (Lycopersicon esculentum cv Ailsa Craig) polygalacturonase genes TAPG1 (LYCes;Pga1;2) and TAPG4 (LYCes; Pga1;5) are abundantly expressed in both abscission zones and the pistils of mature flowers. To further investigate the spatial and temporal expression patterns for these genes, the TAPG gene promoters were ligated to ␤-glucuronidase (GUS) reporter genes and transformed into tomato. GUS expression with both constructs was similar and entirely consistent with the expression patterns of the native gene transcripts. GUS activity was observed in the weakening abscission zones of the leaf petiole, flower and fruit pedicel, flower corolla, and fruit calyx. In leaf petiole and flower pedicel zones this activity was enhanced by ethylene and inhibited by indole-3-acetic acid. On induction of abscission with ethylene, GUS accumulation was much earlier in TAPG4:GUS than in TAPG1:GUS transformants. Moreover, TAPG4:GUS staining appeared to predominate in the vascular bundles relative to surrounding cortex cells whereas TAPG1:GUS was more evenly distributed across the separation layer. Like the native genes, GUS was also expressed in the stigma. Activity was not apparent in pistils until the flowers had opened and was confined to the stigma and style immediately proximal to it. A minimal promoter construct consisting of a 247-bp 5Ј-upstream element from TAPG1 was found to be sufficient to direct GUS expression in both abscission zones and the stigma.

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 (pBACI). 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 pBACI 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 pBACI 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 pBACI or expression of cellulase activity. point (pI 9.5, subsequently referred to as 9.5 cellulase) (6). This 9.5 cellulase was purified and used to prepare cellulase specific antiserum (6). Durbin et al. (6) used a sensitive radioimmune assay to show a close correlation between the appearance of the 9.5 cellulase protein and the onset of abscission. Sexton et al. (27) extended this correlation to show that 9.5 cellulase is predominantly associated with a two to three cell wide separation layer though low levels of activity were also found in adjacent stem and petiolar tissue.Leaf abscission can be induced in the petiole by removal of the subtending leaf blade. Jackson and Osborne (13) Treatment of Plant Materials. Bean (Phaseolus vulgaris cv Red Kidney) plants were grown in a greenhouse and harvested 12 d after germination at which time primary leaves are fully expanded. Explants were prepared by cutting the stem below the cotyledons and then removing the cotyledons. The leaf blades of the primary leaves were removed from explants by cutting 2 cm above the basal pulvinus. The unexpanded trifoliate leaves above the first node were also detached from the explants. Explants were then surface sterilized with a 2 min bath in 0.5% sodium hypochlorite (bleach), rinsed with several volumes of water, and placed upright in styrofoam cups filled with distilled water. Cups containing the explants were placed into a 22 L chamber and the explants exposed to 5 gL/L ethylene in air at a flow rate of 2 L/min. Where indicated, 5 x 1o-6 M IAA in lanolin was applied onto the distal petiolar stump of explants 4 h prior to ethylene treatment. In experim...

No abstract

The changes in the levels of two different isozymes of eellulase (EC 3.2.1.4) have been followed during the abscission of the primary leaves of bean {Phaseolus vulgaris c.v. Red Kidney), using antibodies raised against the 9.5 form of the enzyme. Data from both radioimmune and direct assay show that the 9.5 form of eellulase is undetectable prior to the induction of abscission. After a 12 h lag this isozyme increases in activity, the increase preceding a decrease in integrity of the abscission zone cell walls. The results are consistent not only with the view that this specific isozyme is involved in wall hydrolysis but also with previous data which showed that eellulase is synthesized 'de novo'.The 4.5 isozyme of eellulase is more widely spread throughout the plant, being most active in young tissues. During abscission the activity of this isozyme in the abscission layer falls and consequently it is not thought to be involved directly in the abscission process.

No abstract

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.

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.