Browsing by Author "Denby, Katherine J"
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- ItemOpen AccessActivation of seed-specific genes in leaves and roots of the desiccation tolerant plant, Xerophyta humilis(2008) Walford, Sally-Ann; Illing, Nicola; Farrant, Jill M; Denby, Katherine JThe ability of tissues to survive almost complete loss of cellular water is a trait found throughout the plant kingdom. While this desiccation tolerance is common in seeds of most angiosperms it is rare in their vegetative tissues. Xerophyta humilis (Bak.) Dur and Schintz belongs to a small group of resurrection angiosperms and it possesses the ability to withstand extreme desiccation of greater than 90% in both its seeds and vegetative tissues and return to active metabolism upon rehydration. We have tested the hypothesis that vegetative desiccation tolerance in angiosperms has evolved as an adaptation of seed desiccation tolerance.
- ItemOpen AccessAn investigation into the role of cytosolic free Ca2+ in Salicylic acid mediation of disease resistance in Arabidopsis(2001) Petersen, Lindsay Natalie; Denby, Katherine JSalicylic acid (SA) accumulation upon pathogen attack is a fundamental requirement for the activation of numerous plant defence mechansms. Cytosolic free Ca2+ ([Ca2+]c) is a ubiquitous signalling molecule involved in a host of cellular processes. Using transgenic Arabidopsis thaliana seedlings expressing the Ca2+-sensitive photoprotein aequorin, we previously reported a rapid and transient increase in [Ca2+]c upon application of exogenous SA. We now investigated the characteristics of the SA-induced [Ca2+]c increase and report that the majority of the response is derived from internal stores. It appears likely that SA triggers Ca2+-induced Ca2+-release. Preliminary evidence suggests a role for the SA-induced [Ca2+]c increase in the regulation of NPR1 expression since modulation of the SA-induced [Ca2+]c response with the extracellular Ca2+ chelator BAPTA causes a reduction in NPR1 mRNA levels. We have isolated two putative mutants that are defective in their ability to produce a SA-induced [Ca2+]c increase. Characterisation of these mutants is underway and will prove invaluable in identifying the components or events that cause the SA-induced [Ca2+]c transient, thereby aiding in the understanding of the role of [Ca2+]c in SA-mediated signal transduction.
- ItemOpen AccessDissecting the jasmonate singalling pathway in Arabidopsis thaliana(2008) Wei, Yu-Hung; Denby, Katherine JThe work outlined in this thesis aimed to further our understanding of jasmonate signalling by identifying specific promoter elements and associated transcription factors important for gene regulation by methyl jasmonate. A cDNA encoding a β-glucosidase, PYK10, was previously isolated using the differential display technique in an attempt to isolate methyl jasmonate-induced genes in Arabidopsis thaliana. β-glucosidases catalyse the breakdown of glucosinolates into glucose and sulphate releasing toxic by-products which play a role in plant defence against insects and pathogens.
- ItemOpen AccessGenetic and phenotypic characterisation of an Arabidopsis thaliana jasmonic acid signalling mutant, jam2(2003) Sanderson, Micheline; Denby, Katherine JJasmonic acid (JA) is a plant hormone with diverse functions, ranging from development to stress responses. Moreover, a role for JA in mediating defence against pathogen attack has been established, seemingly specific against necrotrophic pathogens such as Botrytis cinerea. Despite these known roles of JA, it is not known exactly how JA activated downstream responses, such as induced gene expression. To further our understanding of JA signalling, this work aimed to identify new components involved in JA signal transduction. A novel screening method based on lack of anthocyanin accumulation after exogenous application of the methyl ester of JA, methyl jasmonate (MeJA), was employed. A recessive, monogenic mutant, jasmonic acid modified2 (jam2), was isolated from T-DNA activation tagged lines and characterized genetically and phenotypically. jam2 was found not to be T-DNA tagged as the T-DNA segregates independently of the mutation. jam2 is unlikely to be an anthocyanin biosynthetic mutant but shows delayed anthocyanin accumulation after exogenous MeJA treatment. Resistance to MeJA root growth inhibition is a phenotype shared by all JA insensitive mutants. Contrary to this, jam2, like Col-0, exhibits stunted root growth on MeJA. The expression of the antifungal peptide, PDF1.2 can be induced by exogenous MeJA treatment. To assess how PDF1.2 expression was affected in jam2, plants were treated with external liquid and vaporous MeJA. Interestingly, the PDF1.2 expression pattern after MeJA application (liquid or gaseous) was biphasic for Col-0, jam2 and jar1. However, compared to Col-0 and jar1, jam2 appeared to be affected in the first induction peak upon liquid MeJA treatment, whilst in the second after gaseous treatment. PDF1.2 expression can also be seen as a marker for JA-mediated defence responses. Upon infection with B. cinerea, jam2 and jar1 showed intermediate resistance and faster PDF1.2 expression, compared to Col-0 and coil-1. These findings suggest that jam2 is possibly involved in temporal regulation of anthocyanin accumulation and PDF1.2 expression after MeJA application and B. cinerea infection. Therefore, jam2 may define a novel component within the JA signalling pathway and further genetic and phenotypic characterisation could confirm this.
- ItemOpen AccessIncreased resistance to biotrophic pathogens in the Arabidopsis constitutive induced resistance 1 mutant is EDS1 and PAD4-dependent and modulated by environmental temperature(Public Library of Science, 2014) Carstens, Maryke; McCrindle, Tyronne K; Adams, Nicolette; Diener, Anastashia; Guzha, Delroy T; Murray, Shane L; Parker, Jane E; Denby, Katherine J; Ingle, Robert AThe Arabidopsis constitutive induced resistance 1 ( cir1 ) mutant displays salicylic acid (SA)-dependent constitutive expression of defence genes and enhanced resistance to biotrophic pathogens. To further characterise the role of CIR1 in plant immunity we conducted epistasis analyses with two key components of the SA-signalling branch of the defence network, ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We demonstrate that the constitutive defence phenotypes of cir1 require both EDS1 and PAD4, indicating that CIR1 lies upstream of the EDS1-PAD4 regulatory node in the immune signalling network. In light of this finding we examined EDS1 expression in cir1 and observed increased protein, but not mRNA levels in this mutant, suggesting that CIR1 might act as a negative regulator of EDS1 via a post-transcriptional mechanism. Finally, as environmental temperature is known to influence the outcome of plant-pathogen interactions, we analysed cir1 plants grown at 18, 22 or 25°C. We found that susceptibility to Pseudomonas syringae pv. tomato ( Pst ) DC3000 is modulated by temperature in cir1 . Greatest resistance to this pathogen (relative to PR-1:LUC control plants) was observed at 18°C, while at 25°C no difference in susceptibility between cir1 and control plants was apparent. The increase in resistance to Pst DC3000 at 18°C correlated with a stunted growth phenotype, suggesting that activation of defence responses may be enhanced at lower temperatures in the cir1 mutant.
- ItemOpen AccessInvestigating the Role of Cyclic Nucleotide gated channels in Plant- Pathogen Interactions(2009) Adams, Nicolette; Denby, Katherine J
- ItemOpen AccessInvestigation of defence mechanisms against Botrytis cinerea in Arabidopsis thaliana(2005) Adams, Nicolette; Denby, Katherine JDisease resistance in plants has been extensively studied for the past century with many new and exciting results being discovered each year. A plant utilises both preformed and induced defence responses to resist pathogen attack but researchers have focused on dissecting the induced defence response pathway. The complex signal transduction pathway underlying the establishment of resistance to a wide range of pathogen attack is currently being dissected using Arabidopsis thaliana as a model organism. Arabidopsis mutants displaying altered disease resistance response to pathogen infections can help us to get a beUer understanding of the genetiC and molecular basis of the disease resistance pathway. Extensive research has shown that accumulation of 3 signalling molecules are vitally important for establishing a resistance response, as aberrant signalling or accumulation of salicylic acid , ethylene or jasmonic acid `leads to an altered resistance response. Researchers continue to isolate and characterise defence-related mutants to piece together the intricate puzzle of defence-signalling components. A dominant Arabidopsis mutant, constitutive induced resistance 3 (cir3), had been isolated from an ethylmethane sulfonate (EMS) mutagenised transgenic line expressing luciferase under the control of the PR-1 promoter (PR-1
- ItemOpen AccessMicroarray experiments: Considerations for experimental design(Academy of Science of South Africa, 2005) Naidoo, Sanushka; Denby, Katherine J; Berger, Dave KMicroarrays are useful tools to investigate the expression of thousands of genes rapidly. Some researchers remain reluctant to use the technology, however, largely because of its expense. Careful design of a microarray experiment is key to generating cost-effective results. This article explores issues that researchers face when embarking on a microarray experiment for the first time. These include decisions about which microarray platform is available for the organism of interest, the degree of replication (biological and technical) needed and which design (direct or indirect, loop or balanced block) is suitable.
- ItemOpen AccessMolecular characterization of the Arabidopsis thaliana - Botrytis cinerea interaction(2008) Mulema, Joseph Mary K; Denby, Katherine JThis study attempted to characterize at a transcriptional level, the defence responses of Arabidopsis thaliana after infection by Botrytis cinerea, using microarrays. The first microarray experiment focused on profiling Arabidopsis genes induced by B. cinerea over time (temporal) while the second investigated spatial expression of Arabidopsis genes from the point of inoculation. A number of genes were up- and down-regulated specifically at 12 hrs, others at 24 hrs while others were up- and down-regulated at both time points. Similarly, some genes were specifically induced very close to the lesion while others in more distal tissue.
- ItemOpen AccessThe regulation and role of oxidative signal-inducible 1 protein kinase in Arabidopsis thaliana(2007) Petersen, Lindsay Natalie; Denby, Katherine J; Knight, MarcThis study attempted to further characterise OXI1 protein kinase. Confocal microscopy and subcellular fractionation studies revealed a cytosolic localisation pattern for OXI1. Employment of a bioinformatics approach confirmed the induction of OXI1 gene expression in response to a range of AOS generating stimuli. However, the transcriptional increase of OXI1 in response to salinity and heat appears to be of no biological significance since the oxi1 mutant did not display altered tolerance to these two stresses in comparison to wild type.
- ItemRestrictedThe signature of seeds in resurrection plants: a molecular and physiological comparison of desiccation tolerance in seeds and vegetative tissues(Oxford University Press, 2005) Illing, Nicola; Denby, Katherine J; Collett, Helen; Shen, Helen; Farrant, Jill MDesiccation-tolerance in vegetative tissues of angiosperms has a polyphyletic origin and could be due to 1) appropriation of the seed-specific program of gene expression that protects orthodox seeds against desiccation, and/or 2) a sustainable version of the abiotic stress response. We tested these hypotheses by comparing molecular and physiological data from the development of orthodox seeds, the response of desiccation-sensitive plants to abiotic stress, and the response of desiccation-tolerant plants to extreme water loss. Analysis of publicly-available gene expression data of 35 LEA proteins and 68 anti-oxidant enzymes in the desiccation-sensitive Arabidopsis thaliana identified 13 LEAs and 4 anti-oxidants exclusively expressed in seeds. Two (a LEA6 and 1-cys-peroxiredoxin) are not expressed in vegetative tissues in A. thaliana, but have orthologues that are specifically activated in desiccating leaves of Xerophyta humilis. A comparison of antioxidant enzyme activity in two desiccation-sensitive species of Eragrostis with the desiccation-tolerant E. nindensis showed equivalent responses upon initial dehydration, but activity was retained at low water content in E. nindensis only. We propose that these antioxidants are housekeeping enzymes and that they are protected from damage in the desiccation-tolerant species. Sucrose is considered an important protectant against desiccation in orthodox seeds, and we show that sucrose accumulates in drying leaves of E. nindensis, but not in the desiccation-sensitive Eragrostis species. The activation of ‘‘seed-specific’’ desiccation protection mechanisms (sucrose accumulation and expression of LEA6 and 1-cys-peroxiredoxin genes) in the vegetative tissues of desiccation-tolerant plants points towards acquisition of desiccation tolerance from seeds
- ItemOpen AccessUnderstanding the mechanisms of cir1 disease resistance in Arabidopsis thaliana(2008) Carstens, Maryke; Denby, Katherine J; Illing, NicolaPlants have evolved an elaborate and very effective defence system to curb disease caused by pathogen infections. To gain insight into the defence signalling network and defence responses deployed by plants for resistance to pathogens, the defence-related Arabidopsis thaliana mutant cir1 (constitutively induced resistance 1) was further investigated. It was previously shown that cir1 constitutively expresses salicylic acid-, jasmonic acid- and ethylene-dependent defence-related genes and exhibits increased resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato and the virulent oomycete pathogen Hyaloperonospora parasitica Noco2. Through first–pass mapping experiments, it was formerly determined that the CIR1 locus is located on the lower arm of chromosome IV. With the aim of identifying the CIR1 gene, comprehensive genomic mapping of cir1 was conducted in this study. Upon the generation of a suitable mapping population, PCR-based markers were employed to narrow down CIR1 location to 309.10 kb. This region was included in six genomic DNA clones which were tested for complementation of the cir1 mutant. A small region in which CIR1 resides was identified and possible candidate genes within it were investigated. It was established that CIR1 is one of eight annotated genes. This study also assessed which known components of the defence signalling network play a role in cir1-mediated resistance, to establish a possible function of CIR1 in the Arabidopsis defence network. Epistasis analyses were performed between cir1 and the eds1 (enhanced disease susceptibility 1) and pad4 (phytoalexin deficient 4) mutants which regulate the salicylic acid signalling pathway, as well as the coi1 (coronatine-insensitive 1) mutant which functions in the jasmonic acid signalling pathway. The disease resistance profiles of cir1 eds1, cir1 pad4 and cir1 coi1 double mutants to infection by virulent P. syringae and virulent H. parasitica established that coi1, pad4 and eds1 are epistatic to cir1, suggesting that CIR1 is located upstream in the defence signalling network. Through defence-related gene expression profiling, it was found that cir1 simultaneously activates multiple signalling pathways, resulting in the induced expression of many defence-related genes and the increased expression of some of these genes was correlated to cir1’s enhanced resistance to virulent pathogens. Therefore, it appears that CIR1 functions as a negative regulator of the disease resistance signalling network. Furthermore, EDS1 protein accumulation may play a role in cir1-mediated resistance as it was found that cir1 has a stabilizing effect on the EDS1 protein.