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.

The ocs element in the soybean GH2/4 promoter is activated by both active and inactive auxin and salicylic acid analogues

“…1A). Lui and Lam [36] also failed to detect auxin inducibility of a 941 bp CaMV 35S promoter in transgenic tobacco seedlings, and we have observed that our 941 bp CaMV 35S promoter fails to respond to auxins and other agents (e.g., SA) in transgenic tobacco seedlings as well as in carrot protoplasts ( [35,37]; and results presented here). Failure of the CaMV 35S promoter to respond to auxins and other agents in the assay systems described above probably results from the presence of additional elements within this promoter [7] that mask the inducibility of the ocs element.…”

“…Our studies on single G-box-like or hex-like elements [30] in the GH3 promoter (referred to as TGA boxes [27,37]) indicated that, like the animal AP-1 sites, single b-ZIP-binding sites do not confer auxin inducibility (or inducibility by other agents referred to above) to a minimal (i.e., -46) CaMV 35S promoter (Z. Liu and T. Ulmasov, unpublished results). Tandem binding sites for b-ZIP-type proteins appear to be a common occurrence in some plant and animal G S T promoters that are induced by a wide variety of agents, and this suggests that the organization and spacing of these tandem binding sites may be important for the regulation of G S T gene expression (i.e., at least some G S T genes) in both plants and animals.…”

“…Oligonucleotides were synthesized at the University of Missouri DNA Core Facility. The minimal CaMV 35S promoter/GUS reporter gene (p35S-46-TMV-GUS) was made up of the -46 CaMV 35S promoter fused to the tobacco mosaic virus (TMV) ~ translational enhancer and the GUS reporter gene followed by the nos 3' UTR [35,37]. Constructs containing 1, 2, and 4 copies of the ocs element and 4 copies of the S A R E element (i.e., 2 x 37-mer which contains 2 S A R E elements) in the same orientation as in the GH2/4 promoter were tested in transfection assays.…”

“…All constructs used in this study were verified by DNA sequencing. For transient expression assays of GH2/4 promoter-GUS reporter constructs, 10/~g of uncut plasmid DNAs were transfected into 6 x 105 carrot protoplasts obtained from actively growing suspension cell cultures [21,37]. A 941 bp CaMV 35S promoter-luciferase reporter gene was used as an internal standard in all transfection assays ( [5]; provided by Dr John Rogers, University of Missouri, Columbia).…”

An S-RNase promoter from Nicotiana alata functions in transgenic N. alata plants but not Nicotiana tabacum

“…Genes controlled by the 35S promoter are usually expressed most highly in immature and vegetative tissues [2,37,44]. Analyses of many plant genes indicate that spatial, temporal and environmental regulation of gene expression can involve complex interactions between cis-elements, usually located upstream of the coding region [13,19,30,31,45]. In some cases, eis-elements located within exons also contribute to the regulation of gene expression [12,15,32].…”