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 Ϫ50 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.

AINTEGUMENTA negatively regulates age-dependent leaf senescence downstream of AUXIN RESPONSE FACTOR 2 in <i>Arabidopsis thaliana</i>

“…Auxin plays a negative role in leaf senescence by repressing transcription of some genes associated with senescence (Noh and Amasino 1999;Tucker et al 2002). As a member of the auxin response factor (ARF) family, ARF2 also takes part in the auxinmediated control of leaf longevity by repressing auxin responsive gene expression (Ellis et al 2005;Lim et al 2010;Tiwari et al 2003).…”

“…Early studies indicate that auxin acts as a suppressor of leaf senescence by repressing transcription of some genes associated with senescence (Noh and Amasino 1999;Tucker et al 2002). AUXIN RESPONSE FACTOR 2 (ARF2), a member of the auxin response factor (ARF) family, represses auxin responsive gene expression (Tiwari et al 2003).…”

AUXIN RESPONSE FACTOR1andAUXIN RESPONSE FACTOR2regulate senescence and floral organ abscission inArabidopsis thaliana

“…Plant hormones that influence senescence include ethylene, abscisic acid and jasmonates, which can induce senescence; and auxin, cytokinin and gibberellins, which can play a role in its suppression (Lim et al, 2003). Auxin represses transcription of some genes whose expression is correlated with senescence and/or abscission (Noh and Amasino, 1999;Hong et al, 2000;Tucker et al, 2002).…”

Citrus biotechnology: Achievements, limitations and future directions

“…Transient expression systems are designed for short-term studies of gene function and regulation (Barandiaran et al, 1998, Ferrer et al, 2000 and are advantageous over other protocols in being rapid, inexpensive and uncomplicated procedures. This type of protocol is particularly beneficial and uncomplicated, as it does not require expensive apparatus or tedious and timeconsuming methods for assessment of the activity of transgenes (Tucker et al, 2002;Hoffmann et al, 2004). A significant, yet simple method for transient expression of genes in fleshy fruits via Agrobacterium-mediated transformation was proposed by Spolaore et al (2001), wherein they transformed intact ripe and fleshy fruits of apple, strawberry, orange, tomato and peach by directly injecting them with a syringe containing Agrobacterium suspension.…”

“…Being a transient gene expression system, it offers an additional advantage of studying promoter strength and function in a short duration of time as regeneration of transformed cells into plants is not required (Tucker et al, 2002;Hoffmann et al, 2004). Moreover, this system is especially valuable in the study of species that are recalcitrant to transformation or that bear fruit after a long duration of time post transformation.…”