The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase

Abstract

Bean leaf abscission (organ separation) correlates with the de novo accumulation of a pI9.5 cellulase and its mRNA. Overlapping genomic clones encoding the bean abscission cellulase (BAC) were isolated and partially sequenced. In addition, a genomic clone for a soybean abscission cellulase (SAC) was identified and the sequence compared to the BAC genomic sequence. Two 5'-upstream regions are particularly well conserved in the two sequences. Of special interest here is the region between -1 and -200 in the BAC promoter which is highly conserved in the SAC gene. Particle gun bombardment with a BAC promoter construct containing 210 bp of BAC sequence 5' to the transcription start site was sufficient to drive abscission-specific and ethylene and auxin-regulated transient expression in bean. In addition to the transient expression assay, expression was examined in stably transformed tomato. A similar -210 bp BAC promoter construct supported a low level of ethylene-inducible reporter gene expression in tomato leaf abscission zones and adjacent petioles but not in ethylene-treated stem tissue or fruit. Expression from the -210 promoter in tomato abscission zones was inhibited by silver thiosulfate, an ethylene action inhibitor, and was partially inhibited by treatment with auxin.

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).…”

“…Earlier studies demonstrated that a 210-bp proximal BAC promoter retained tissue specificity and hormonal regulation similar to a 2.9-kb BAC promoter, but at a much reduced level of expression (Koehler et al, 1996). To characterize specific elements within the proximal BAC promoter, sitedirected mutagenesis was used to generate a series of 15 substitutions of 10 bp each between Ϫ172 and Ϫ23 bp (relative to the start of transcription) in a fulllength 2.9-kb BAC promoter (BKm3; Fig.…”

“…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).…”

“…To further enhance our understanding of these processes, we embarked on a study of the BAC promoter to identify cis-elements and trans-acting factors that regulate abscission-specific expression. Earlier studies with the BAC promoter in stably transformed tomato (Lycopersicon esculentum) and transient expression assays in bean explants demon-strated that 210 bp of the proximal 5Ј-upstream BAC sequence was sufficient for ethylene-and auxinregulated expression (Koehler et al, 1996). In bean explants, expression from this minimal BAC promoter was restricted primarily to the AZ.…”

Temporal and spatial expression of mRNAs encoding pathogenesis‐related proteins during ethylene‐promoted leaflet abscission in Sambucus nigra*

“…One apptoach (o studying this has been to isolate the genes encoding certain key enzynies, such as cell wall hydtolases, and analyse (he protTioters regulating their expiession (Koehler et al 1996). However, the exptession of additional geties tnay conttibute, in a critical way, to the abscission process and their identification will be itiiportant in furthering our understanding of how the ptocess is regulated.…”