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

Abstract

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-bp mutation includes a TGA-type basic leucine zipper (bZIP) motif. Tandem ligation of three 18-bp 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.

Figure 1

Construction of 10 bp poly(A⁺) substitutions and bZIP mutation in the 2.9-kb BAC promoter (BKm3) and their transient expression in bean explants. A, Construct design and graphical representation of substitutions within the proximal BAC promoter region. B, Transient expression results. Expression levels are the ratios of luciferase to GUS activity normalized to the ratio for the external control that consists of a native BAC promoter in BKm3 shot into AZ explants exposed to ethylene for 48 h. Treatments were 1 μL L⁻¹ ethylene in air, auxin as 10⁻⁴ m 2,4-dichlorophenoxyacetic acid (2,4-D), and 5,000 μL L⁻¹ 2,5-norbornadiene (NBD). Each mean and se represents six or more replicate plates containing nine AZ explants or five stems and 10 petiole explants.

Figure 2

Transient expression characteristics of two minimal CaMV 35S promoters (−50 and −90) fused to three tandem 18-bp repeats of the native, mutant, or reverse complement of the Z-BAC element. A, Luciferase constructs with a minimal −50 and −90 35S promoter fused to tandem repeats of the native Z-BAC element (zf), mutant Z-BAC element (zfm), and reverse complement Z-BAC element (zr). Relative transient expression of the −50 35S (B) and −90 35S (C) minimal constructs in bean AZ and stem explants. Ethylene, auxin, and NBD treatments are as described in Figure 1. Unmodified ratios for luciferase to GUS activity are shown to allow a direct comparison of the expression levels from the −50 and −90 35S minimal constructs. Each mean and se represents four replicate plates containing nine AZs or nine stem explants.

Figure 3

Transient expression characteristics of the e35S::GUS construct in AZs and stems. A, Construct design. B, Absolute expression levels of GUS activity in AZs and stems for all the replicates used in the normalized data displayed in Figure 2. Each mean and se represents 30, 20, and 18 replicate plates exposed to ethylene, NBD, or ethylene plus auxin, respectively, as described in Figure 2.

Figure 4

EMSA for binding of bean AZ transcription factors to a 24-bp Z-BAC+ element. A, Sequence for wild-type (WT) and mutant (Mu) oligonucleotides. B, Transcription factors tested were in vitro transcribed and translated from three full-length TGA-type bZIP cDNAs isolated from a bean AZ cDNA library and competition experiments included 100-fold excess of unlabeled wild-type (WT) Z-BAC+ element or mutant Z-BAC+ element (Mu) or no unlabeled DNA added (−). First lane, Incubation where no protein or competitive DNA was added.

Figure 5

RNA-blot analysis for the expression patterns of three bean TGA-type bZIP transcription factors and the BAC gene. Several replica gels and blots were prepared from the same batch of RNA and hybridized separately with labeled cDNA probes. Leaf AZ (LAZ) or stem explants were collected and exposed to 25 μL L⁻¹ ethylene, 10⁻⁵ m indole-3-acetic acid (IAA), and/or 5,000 μL L⁻¹ NBD for 0, 24, or 48 h. Leaf (L) and root (R) RNA were collected from fresh untreated organs (0 h).