Bean Abscission Cellulase

Abstract

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...

Functional Analysis of Regulatory Elements in the Gene Promoter for an Abscission-Specific Cellulase from Bean and Isolation, Expression, and Binding Affinity of Three TGA-Type Basic Leucine Zipper Transcription Factors

“…In contrast, mutant Ϫ33 immediately upstream from the TATA box, displayed a 20% increase in expression, and mutant Ϫ113 displayed a 40% increase in expression in ethylene-treated AZ. Auxin (10 Ϫ4 m 2,4-D), which strongly inhibits abscission and cellulase gene expression even in the presence of high concentrations of ethylene (Tucker et al, 1988;Koehler et al, 1996), inhibited expression from the native BKm3 and most of the mutant constructs by 40% to 50% (Fig. 1B).…”

“…Although the control of abscission is not identical for all plant parts, a common pattern for the regulation of abscission is that ethylene induces and enhances the process, whereas auxin strongly inhibits it (Sexton and Roberts, 1982). Expression of the bean abscission cellulase (BAC) mRNA correlates precisely with the onset of abscission and its regulation by ethylene and auxin (Tucker et al, 1988). Moreover, after ethylene treatment to induce abscission, expression of the BAC mRNA is limited to a depth of one or two cells on either side of the fracture plane in nonvascular tissue and up to 3 mm distal to the fracture plane in cells within the vascular bundle.…”

“…One of the first model systems used for abscission research was bean (Phaseolus vulgaris). It was in bean leaf AZs that the first abscission-specific hydrolase, ␤-1,4-endoglucanase, was characterized (Lewis and Varner, 1970), purified (Koehler et al, 1980), and the cDNA (Tucker et al, 1988) and genomic (Koehler et al, 1996) clones identified. Although the control of abscission is not identical for all plant parts, a common pattern for the regulation of abscission is that ethylene induces and enhances the process, whereas auxin strongly inhibits it (Sexton and Roberts, 1982).…”

Identification and Kinetics of Accumulation of Proteins Induced by Ethylene in Bean Abscission Zones

“…In bean, a cellulase, referred to as 9.5 cellulase, has been implicated in abscission. This cellulase is induced by ethylene and accumulates to high levels in the abscission zone (25). The increase in activity of 9.5 cellulase has been correlated with the decrease in break-strength in the separation layer (9).…”

“…In fact, addition of purified 9.5 cellulase to plant cells growing in tissue culture cannot form protoplasts unless cellulase is added in combination with fungal pectinase (15). Also, the increase in cellulase message and protein, after the addition of ethylene, is preceded by a long lag period of 24 to 30 h (25). Thus, it is possible that cellulase is preceded by the hydrolytic action of other enzymes.…”