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.

Abscisic acid regulation of plant defence responses during pathogen attack

The plant hormone, abscisic acid (ABA), has previously been shown to have an impact on the resistance or susceptibility of plants to pathogens. In this thesis, it was shown that ABA had a regulatory effect on an extensive array of plant defence responses in three different plant and pathogen interaction combinations as well as following the application of an abiotic elicitor. In unique studies using ABA deficient mutants of Arabidopsis, exogenous ABA addition or ABA biosynthesis inhibitor application and simulated drought stress, ABA was shown to have a profound effect on the outcome of interactions between plants and pathogens of differing lifestyles and from different kingdoms. The systems used included a model plant and an important agricultural species: Arabidopsis thaliana (Arabidopsis) and Peronospora parasitica (a biotrophic Oomycete pathogen), Arabidopsis and Pseudomonas syringae pathovar tomato (a biotrophic bacterial pathogen) and an unrelated plant species, soybean (Glycine max) and Phytophthora sojae (a hemibiotrophic Oomycete pathogen), Generally, a higher than basal endogenous ABA concentration within plant tissues at the time of avirulent pathogen inoculation, caused an interaction shift towards what phenotypically resembled susceptibility. Conversely, a lower than basal endogenous ABA concentration in plants inoculated with a virulent pathogen caused a shift towards resistance. An extensive suppressive effect of ABA on defence responses was revealed by a range of techniques that included histochemical, biochemical and molecular approaches. A universal effect of ABA on suppression or induction of the phenylpropanoid pathway via regulation of the key entry point gene, phenylalanine ammonia-lyase (PAL), when stimulated by biotic or abiotic elicitors was shown. ABA also influenced a wide variety of other defence-related components such as: the development of a hypersensitive response (HR), the accumulation of the reactive oxyden species, hydrogen peroxide and the cell wall strengthening compounds lignin and callose, accumulation of SA and the phytoalexin, glyceollin and the transcription of the SA-dependent pathogenesis- related gene (PR-1). The near genome-wide microarray gene expression analysis of an ABA induced susceptible interaction also revealed an yet unprecedented insight into the great diversity of defence responses that were influenced by ABA that included: disease resistance like proteins, antimicrobial proteins as well as phenylpropanoid and tryptophan pathway enzymes. Subtle differences were found in the number and type of defence responses that were regulated by ABA in each type of plant and pathogen interaction that was studied. This thesis has clearly identified in plant/pathogen interactions previously unknown and important roles for ABA in the regulation of many defence responses.

Evaluation of heat stress indices using physiological comparisons in an alumina refinery in a sub-tropical climate

The production of alumina involves the use of a process known as the Bayer process. This method involves the digestion of raw bauxite in sodium hydroxide at temperatures around 250°C. The resultant pregnant liquor then goes through a number of filtering and precipitation processes to obtain the aluminium oxide crystals which are then calcined to obtain the final product. The plant is situated in a sub tropical climate in Northern Australia and this combined with the hot nature of the process results in a potential for heat related illnesses to develop. When assessing a work environment for heat stress a heat stress index is often employed as a guideline and to date the Wet Bulb Globe Temperature (WBGT) has been the recommended index. There have been concerns over the past that the WBGT is not suited to the Northern Australian climate and in fact studies in other countries have suggested this is the case. This study was undertaken in the alumina plant situated in Gladstone Queensland to assess if WBGT was in fact the most suitable index for use or if another was more applicable. To this end three indices, Wet Bulb Globe Temperature (WBGT), Heat Stress Index (HSI) and Required Sweat Rate (SWreq) were compared and assessed using physiological monitoring of heart rate and surrogate core temperature. A number of different jobs and locations around the plant were investigated utilising personal and environmental monitoring equipment. These results were then collated and analysed using a computer program written as part of the study for the manipulation of the environmental data . Physiological assessment was carried out using methods approved by international bodies such as National Institute for Occupational Safety & Health (NIOSH) and International Standards Organisation (ISO) and incorporated the use of a ‘Physiological Factor’ developed to enable the comparison of predicted allowable exposure times and strain on the individual. Results indicated that of the three indices tested, Required Sweat Rate was found to be the most suitable for the climate and in the environment of interest. The WBGT system was suitable in areas in the moderate temperature range (ie 28 to 32°C) but had some deficiencies above this temperature or where the relative humidity exceeded approximately 80%. It was however suitable as a first estimate or first line indicator. HSI over-estimated the physiological strain in situations of high temperatures, low air flows and exaggerated the benefit of artificial air flows on the worker in certain environments ie. fans.

Increased Biomass, Seed Yield and Stress Tolerance Is Conferred in Arabidopsis by a Novel Enzyme from the Resurrection Grass Sporobolus stapfianus That Glycosylates the Strigolactone Analogue GR24

Isolation of gene transcripts from desiccated leaf tissues of the resurrection grass, Sporobolus stapfianus, resulted in the identification of a gene, SDG8i, encoding a Group 1 glycosyltransferase (UGT). Here, we examine the effects of introducing this gene, under control of the CaMV35S promoter, into the model plant Arabidopsis thaliana. Results show that Arabidopsis plants constitutively over-expressing SDG8i exhibit enhanced growth, reduced senescence, cold tolerance and a substantial improvement in protoplasmic drought tolerance. We hypothesise that expression of SDG8i in Arabidopsis negatively affects the bioactivity of metabolite/s that mediate/s environmentally-induced repression of cell division and expansion, both during normal development and in response to stress. The phenotype of transgenic plants over-expressing SDG8i suggests modulation in activities of both growth- and stress-related hormones. Plants overexpressing the UGT show evidence of elevated auxin levels, with the enzyme acting downstream of ABA to reduce drought-induced senescence. Analysis of the in vitro activity of the UGT recombinant protein product demonstrates that SDG8i can glycosylate the synthetic strigolactone analogue GR24, evoking a link with strigolactone-related processes in vivo. The large improvements observed in survival of transgenic Arabidopsis plants under cold-, salt- and drought-stress, as well as the substantial increases in growth rate and seed yield under non-stress conditions, indicates that overexpression of SDG8i in crop plants may provide a novel means of increasing plant productivity.

Hyperaccumulation of zinc by Noccaea caerulescens results in a cascade of stress responses and changes in the elemental profile

Noccaea caerulescens (J. & C. Presl) F. K. Meyer is a metal hyperaccumulating plant which can accumulate more than 2% zinc (Zn) dry tissue mass in its aerial tissues. At this concentration Zn is toxic to most plants due to inhibition of enzyme function, oxidative damage and mineral deficiencies. In this study the elemental and metabolite profiles of N. caerulescens plants grown in four different Zn concentrations were measured. This revealed broad changes in the metabolite and elemental profiles with the hyperaccumulation of Zn. The Zn treated plants exhibited no typical signs of stress such as chlorosis or reduced biomass, however, a range of metabolic stress responses, such as the modification of galactolipids and the major membrane lipids of plastids, and increases in oxylipins, which are precursors to the signalling molecules jasmonic and abscisic acids, as well as the increased synthesis of glucosinolates, was observed. Increases in particular organic acids and the ubiquitous metal cation chelator nicotianamine were also observed. The small molecule metabolite changes observed, however, did not account for the extreme Zn concentrations in the leaf tissue showing that the increase in nicotianamine production most likely negates Fe deficiency. The elemental analyses also revealed significant changes in other essential micronutrients, in particular, significantly lower Mn concentrations in the high Zn accumulating plants, yet higher Fe concentrations. This comprehensive elemental and metabolite analysis revealed novel metabolite responses to Zn and offers evidence against organic acids as metal-storage ligands in N. caerulescens. © 2014 The Royal Society of Chemistry.