Soybean GH3 Promoter Contains Multiple Auxin-Inducible Elements

Abstract

The soybean GH3 gene is transcriptionally induced in a wide variety of tissues and organs within minutes after auxin application. To determine the sequence elements that confer auxin inducibility to the GH3 promoter, we used gel mobility shift assays, methylation interference, deletion analysis, linker scanning, site-directed mutagenesis, and gain-of-function analysis with a minimal cauliflower mosaic virus 35S promoter. We identified at least three sequence elements within the GH3 promoter that are auxin inducible and can function independently of one another. Two of these elements are found in a 76-bp fragment, and these consist of two independent 25- and 32-bp auxin-inducible elements. Both of these 25- and 32-bp auxin-inducible elements contain the sequence TGTCTC just upstream of an AATAAG. An additional auxin-inducible element was found upstream of the 76-bp auxin-inducible fragment; this can function independently of the 76-bp fragment. Two TGA-box or Hex-like elements (TGACGTAA and TGACGTGGC) in the promoter, which are strong binding sites for proteins in plant nuclear extracts, may also elevate the level of auxin inducibility of the GH3 promoter. The multiple auxin-inducible elements within the GH3 promoter contribute incrementally to the overall level of auxin induction observed with this promoter.

STF1 is a novel TGACG‐binding factor with a zinc‐finger motif and a bZIP domain which heterodimerizes with GBF proteins

“…Two major classes of plant cis regulatory elements characterized to date that contribute to transcriptional regulation upon diverse stimuli are the TGACG-motif and the G-box element, carrying an ACGT core sequence Giuliano et al, 1988;Menkens et al, 1995;SchulzeLefert et al, 1989;Weisshaar et al, 1991). The TGACGmotif and variations thereof are referred to collectively as C-box (nnGACGTCnn) elements (Izawa et al, 1993) and are found in the promoters of a variety of plant genes including the cauliflower mosaic virus (CaMV) 35S promoter (the as-1 element) (Benfey and Chua, 1990;Lam et al, 1989), the enhancers of the nopaline and octopine synthase genes (the nos and ocs elements, respectively) (Bouchez et al, 1989;Feltkamp et al, 1994;Lam et al, 1990), the wheat histone H3 promoter (the hexamer or Hex element) (Mikami et al, 1989), genes regulated by auxin such as GH3 and GmAux28 (Liu et al, 1994;Nagao et al, 1993), and cell wall protein genes (Hong et al, 1987). The G-box (CACGTG) is also a major class of ubiquitous regulatory DNA elements found in plant genomes, a representative of which is the light-regulated RuBP carboxylase small subunit (RBCS) gene (review by Menkens et al, 1995).…”

Auxin action and auxin‐binding proteins

“…Within two of tbe auxin-responsive elements (termed DI and D4: Table 3) there are two sets of conserved sequence, one of which (TGTCTC) is also present in tbe NDE region of tbe SAUR genes and is similar to the 5' end of motif A oi PS-IAA4/5. Liu et al (1994) also point out that a sequence just 3' to motif A (not shown in Table 3) is similar to part of their DI sequence.…”

“…Nagao et al, Nagao et al, 1993 McClure et al, Nagao et al, 1993Nagao et al, 1993Liu et al, 1994Liu et al, 1994 * Conserved sequence elements shown in bold: note similarity to motif A.…”

“…All auxinregulated genes of elongating tissue characterized so far contain a motif A-like element, and this is particularly reassuring, but it also highlights the fact tbat tbe GST gene family does not sbare tbis motif. The gene GH3 wbich is induced by auxin in both dividing and elongating tissue, does contain motif A (Table 3) but this part of tbe GH3 promoter was not found to confer auxin inducibility (although it might act as an enhancer; Liu et al, 1994). Together with tbe observation that PS-IAA6 was no longer induced by auxin in pea protoplasts (Ballas et al, 1993) this suggests, perhaps, tbat tratiscriptional activation of some or all of tbese genes is regulated by diverse (rather than single) transcriptional factors.…”

“…Recent promoter analysis of the GH3 gene bas indicated that here too there are multiple response elements (Liu et al, 1994). Again, many complementary techniques were used.…”