A PAL1 gene promoter-green fluorescent protein reporter system to analyse defence responses in live cells of Arabidopsis thaliana

Arabidopsis thaliana ecotype Columbia-0 was transformed with a green fluorescent protein (GFP) gene under control of a phenylalanine ammonia-lyase (PAL) promoter. PAL is a key enzyme of the phenylpropanoid pathway and is induced to high levels during plant stress. Constitutive expression of PAL1 promoter-controlled GFP occurred in vascular tissues within stems, leaves and roots and in developing flowers. PAL1 promoter–GFP expression was examined in leaves of transgenic plants subjected to an abiotic elicitor, mechanical wounding or to inoculation with the pathogens Pseudomonas syringae pv. tomato or Peronospora parasitica. Wounding of leaves and treatment with an abiotic elicitor and compatible interactions produced low to moderate levels of GFP. However, in incompatible interactions there were high levels of GFP produced. In incompatible interactions, the intensity of GFP fluorescence was similar to that produced in transgenic plants expressing GFP driven by the CaMV promoter. The bright green fluorescence produced in live cells and tissues was readily visualised using conventional fluorescence microscopy and was quantified using spectroflourometry. This is the first report of the use of GFP as a reporter of defence gene activation against pathogens. It has several advantages over other reporter genes including real time analysis of gene expression and visualisation of defence gene activation in a non-invasive manner.

Hormonal regulation of platypus Beta-lactoglobulin and monotreme lactation protein genes

Endocrine regulation of milk protein gene expression in marsupials and eutherians is well studied. However, the evolution of this complex regulation that began with monotremes is unknown. Monotremes represent the oldest lineage of extant mammals and the endocrine regulation of lactation in these mammals has not been investigated. Here we characterised the proximal promoter and hormonal regulation of two platypus milk protein genes, Beta-lactoglobulin (BLG), a whey protein and monotreme lactation protein (MLP), a monotreme specific milk protein, using in vitro reporter assays and a bovine mammary epithelial cell line (BME-UV1). Insulin and dexamethasone alone provided partial induction of MLP, while the combination of insulin, dexamethasone and prolactin was required for maximal induction. Partial induction of BLG was achieved by insulin, dexamethasone and prolactin alone, with maximal induction using all three hormones. Platypus MLP and BLG core promoter regions comprised transcription factor binding sites (e.g. STAT5, NF-1 and C/EBPα) that were conserved in marsupial and eutherian lineages that regulate caseins and whey protein gene expression. Our analysis suggests that insulin, dexamethasone and/or prolactin alone can regulate the platypus MLP and BLG gene expression, unlike those of therian lineage. The induction of platypus milk protein genes by lactogenic hormones suggests they originated before the divergence of marsupial and eutherians.