Characterisation of cis-elements in the GST-27 promoter of Zea mays.
thesisposted on 19.11.2015, 09:07 by Hilary Morna. Sheppard
The ability to control the expression of selected genes has many applications both in industry and academic research. The 27 kDa subunit of the glutathione S-transferase II (GST-27) gene in maize is not transcribed in the aerial organs of maize during normal growth and development. However, it can be induced by treatment with herbicide safeners (e.g. dichlormid) resulting in high levels of expression. Since the promoter of this gene may be useful as a tool to control the expression of other genes, we have investigated the molecular basis of its induction by the identification of the cis-acting promoter elements involved. Safeners also induce the production of enzymes and substrates which are involved in a plant's defense against pathogen attack and in combating oxidative stress. Thus, information relating to GST-27 induction may also add to our knowledge relating to these other responses. Deletion analysis of the GST-27 promoter was carried out by transient expression of GST- 27::GUS transcriptional fusions in Black Mexican Sweet (BMS) maize cells. These experiments indicated that a 378 base pair region of the promoter can confer safener-inducibility on GUS expression. This region of the promoter was in vivo footprinted and four putative safener-responsive elements were thus identified (regions in which G residues are protected when the gene is expressed). These elements are similar to each other and to an ethylene responsive element in the GST1 gene of carnation. Electrophoresis mobility-shift assays indicated that one or more nuclear proteins from maize leaves specifically interact with all of the elements. These elements were mutated in GST-27::GUS constructs, which were tested in transient assays in BMS cells in order to obtain direct evidence for their involvement in safener-dependent transcription. The transient assay technique was, however, found to be insufficiently sensitive for this purpose. The GST-27::GUS constructs containing mutations of the putative elements were therefore tested in transgenic tobacco. The 570 base pairs of the wild type GST-27 promoter upstream of the transcription start point was found to retain inducibility in transgenic tobacco plants, but a 378 base pair GST-27::GUS transcriptional fusion (which was inducible in BMS maize cell lines) was not inducible when tested in tobacco. In order to test for loss of function, mutations of the putative safener-responsive elements were introduced into the 378 base pair truncated promoter; in order to test for a gain of function the putative elements were fused to the -60 and -90 35S cauliflower mosaic virus minimal promoters and tested in stable tobacco transformants. These experiments did not, however, prove that these elements play a role in safener-dependent transcription. Surprisingly, the -90 minimal promoter itself was found to be responsive to safener. A previously characterised cis-element, activation sequence-1 (as-1), is contained within the -90 minimal promoter and may account for its inducibility; a similar element is present in the GST-27 promoter but its role in safener-dependent transcription, if any, is unclear.