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

Site-directed mutagenesis was used to identify cis-acting elements that control hormonal and abscission-specific expression of the bean (Phaseolus vulgaris) abscission cellulase (BAC) promoter. Auxin inhibition of BAC promoter expression is at least in part controlled by a negatively regulated element and ethylene induction by a positively regulated element. One of a series of 15 different 10-bp mutations created in a 2.9-kb BAC promoter reduced reporter gene expression by 60%. The native sequence for this 10-by mutation includes a TGA-type basic leucine zipper (bZIP) motif. Tandem ligation of three 18-by BAC elements (Z-BAC), which includes the bZIP motif to a minimal -50 35S cauliflower mosaic virus promoter, enhanced expression in abscission zones (AZs) 13-fold over that of the minimal promoter alone. The native forward orientation of the Z-BAC elements was essential for high expression levels. Expression of the Z-BAC minimal construct was 3-fold greater in AZ than stems when compared with the expression levels of an internal control with an enhanced 35S cauliflower mosaic virus promoter. Polymerase chain reaction was used to identify three TGA-type bZIP transcription factors in an AZ cDNA library. One of these factors was of the class I type and two of the class II type. RNA-blot analysis was completed for these genes and electrophoretic mobility shift assays used to confirm their binding to the Z-BAC element. Electrophoretic mobility shift assay-binding affinity was greatest for the class I TGA-type bZIP factor. The results indicate a complex interaction of negative and positive regulating transcription factors that control BAC gene expression.