Comparing suppression subtractive hybridization and bioinformatics approaches for analyzing functional gene expression in Arabidopsis thaliana following a heat shock treatment
- Authors: Bhamjee, Rabia Ahmid
- Date: 2012-05-03
- Subjects: Arabidopsis thaliana , Hybridization , Bioinformatics , Gene expression , Plant gene expression , Plant defenses , Heat shock proteins , Plants, Effect of heat on
- Type: Thesis
- Identifier: uj:2254 , http://hdl.handle.net/10210/4692
- Description: M.Sc. , Since plants are stationary, their immune systems have adapted to their environments to enable them to overcome or respond appropriately to various environmental, physical and physiological stresses that they may encounter by developing complicated defense mechanisms. The plant defense response activates complex biochemical and structural changes in plant cells. Heat stress per se, appears to be a priority stress response in plants, and increased disease susceptibility may be a result of this response. In this study, altered gene expression levels mediated by a heat treatment in Arabidopsis thaliana seedlings were analyzed. Seedlings were exposed to a heat stress of 42C for 30 minutes, followed by a 2.5 hour recovery period at 25ºC. RNA that was isolated from the heat stress treated plants as well as control plants (untreated) was used to perform suppression subtractive hybridization (SSH) in order to obtain a forward and a reverse DNA library. The forward SSH library represented the genes that were up-regulated due to the heat shock and the reverse SSH library represented the down-regulated genes. Sequenced clones from these libraries were BLAST against the A. thaliana genome using the Genbank database and the Accession numbers retrieved were then used for Bioinformatics analysis to obtain functionality of the genes found. The bioinformatics tools used were TAIR tools, DAG graphs and FatiGO and genes were categorized into the biological processes, molecular functions and cellular components. The TAIR tools and FatiGO were then used to analyze microarray data obtained for a similar study, in order to compare the genes identified with SSH. The genes coding for photosystem IID, serine-type peptidase, phospholipase D α, a S-locus protein kinase, regulator of chromosome condensation (RCC1) and Glucose-6-phosphate translocator are prominently up-regulated whilst other genes encoding photosystem I, plastocyanin-like mavicyanin, carbohydrate trans-membrane transporter MSS1, zinc finger C3HC4 ring family protein, ubiquitin conjugating enzyme 35 (UBC35) and integral membrane family proteins are significantly down-regulated. The FatiGO results helped to assign functionality to the genes that were found. For the SSH forward library, the cellular protein metabolic pathway was the most highly expressed term (19.21%), whereas in the microarray data, the term „positive regulation of response to stimulus‟ and membrane disassembly had a 100% expression. The reverse SSH data (down-regulation) found phosphate metabolic process as the most highly expressed term with an expression of 44.36% ix and the microarray data (negative fold-change) found the term photorespiration to be the most highly expressed with 93.54% expression. These high levels of negative expression indicate the down-regulation of these processes in the cell during heat shock. From these results it can be assumed that at the onset of a heat stress, the plant‟s immediate response is to activate pathways of regulation as a response to the stimulus as a self-protection mechanism, and repress other pathways such as photorespiration in order to preserve its energy such as ATP. These findings suggest that the plant is well equipped to overcome stress in its environment by activation/repression of specific organelles and pathways in the system, in order to maintain its equilibrium. Studies such as these can prove to be helpful to solve the interesting question of how a plant overcomes various environmental stresses in order to prevent disease susceptibility.
- Full Text:
- Authors: Bhamjee, Rabia Ahmid
- Date: 2012-05-03
- Subjects: Arabidopsis thaliana , Hybridization , Bioinformatics , Gene expression , Plant gene expression , Plant defenses , Heat shock proteins , Plants, Effect of heat on
- Type: Thesis
- Identifier: uj:2254 , http://hdl.handle.net/10210/4692
- Description: M.Sc. , Since plants are stationary, their immune systems have adapted to their environments to enable them to overcome or respond appropriately to various environmental, physical and physiological stresses that they may encounter by developing complicated defense mechanisms. The plant defense response activates complex biochemical and structural changes in plant cells. Heat stress per se, appears to be a priority stress response in plants, and increased disease susceptibility may be a result of this response. In this study, altered gene expression levels mediated by a heat treatment in Arabidopsis thaliana seedlings were analyzed. Seedlings were exposed to a heat stress of 42C for 30 minutes, followed by a 2.5 hour recovery period at 25ºC. RNA that was isolated from the heat stress treated plants as well as control plants (untreated) was used to perform suppression subtractive hybridization (SSH) in order to obtain a forward and a reverse DNA library. The forward SSH library represented the genes that were up-regulated due to the heat shock and the reverse SSH library represented the down-regulated genes. Sequenced clones from these libraries were BLAST against the A. thaliana genome using the Genbank database and the Accession numbers retrieved were then used for Bioinformatics analysis to obtain functionality of the genes found. The bioinformatics tools used were TAIR tools, DAG graphs and FatiGO and genes were categorized into the biological processes, molecular functions and cellular components. The TAIR tools and FatiGO were then used to analyze microarray data obtained for a similar study, in order to compare the genes identified with SSH. The genes coding for photosystem IID, serine-type peptidase, phospholipase D α, a S-locus protein kinase, regulator of chromosome condensation (RCC1) and Glucose-6-phosphate translocator are prominently up-regulated whilst other genes encoding photosystem I, plastocyanin-like mavicyanin, carbohydrate trans-membrane transporter MSS1, zinc finger C3HC4 ring family protein, ubiquitin conjugating enzyme 35 (UBC35) and integral membrane family proteins are significantly down-regulated. The FatiGO results helped to assign functionality to the genes that were found. For the SSH forward library, the cellular protein metabolic pathway was the most highly expressed term (19.21%), whereas in the microarray data, the term „positive regulation of response to stimulus‟ and membrane disassembly had a 100% expression. The reverse SSH data (down-regulation) found phosphate metabolic process as the most highly expressed term with an expression of 44.36% ix and the microarray data (negative fold-change) found the term photorespiration to be the most highly expressed with 93.54% expression. These high levels of negative expression indicate the down-regulation of these processes in the cell during heat shock. From these results it can be assumed that at the onset of a heat stress, the plant‟s immediate response is to activate pathways of regulation as a response to the stimulus as a self-protection mechanism, and repress other pathways such as photorespiration in order to preserve its energy such as ATP. These findings suggest that the plant is well equipped to overcome stress in its environment by activation/repression of specific organelles and pathways in the system, in order to maintain its equilibrium. Studies such as these can prove to be helpful to solve the interesting question of how a plant overcomes various environmental stresses in order to prevent disease susceptibility.
- Full Text:
In silico analysis of cis elements and expression analysis of selected LPS-responsive RLK genes from Arabidopsis thaliana
- Authors: New, Sherrie-Ann
- Date: 2013-07-29
- Subjects: Plant defenses , Plant-pathogen relationships , Arabidopsis thaliana - Disease and pest resistance
- Type: Thesis
- Identifier: uj:7708 , http://hdl.handle.net/10210/8573
- Description: M.Sc. (Biochemistry) , Our comprehension of pathogen perception and defense response mechanisms that play key roles in the resistance of plants against pathogen attack have progressed substantially within the recent years. Recognizing the molecular mechanisms involved in pathogen perception is the basis of understanding the signalling networks that are involved, including the transcriptional regulation of plant defense genes. This has proven to be a great challenge in plant pathology and, as such, has attracted much attention. The receptor-like kinases (RLKs) constitute one of the largest classes of plant defense genes in Arabidopsis thaliana, and contains, inter alia, the well-known leucine-rich repeats-RLKs (LRR-RLK), as well as the S-domain receptor-like kinases (SD-RLKs) that have been shown to be involved in pathogen perception and not only self-incompatibility (SI) as originally discovered. Some members of these RLKs are able to detect pattern-associated molecular patterns (PAMPs), which are conserved pathogen-derived molecules, and trigger a battery of basal defense responses. The transcriptional activation and expression levels of RLKs are dependent on the variation in promoter architecture as a result of the number, location, order and class of cis-elements found in a promoter sequence. It is hypothesized that candidate RLK genes involved in PAMP surveillance are triggered and transcriptionally regulated in response to perception of PAMPs, and that the intensity of response is relative to the promoter architecture. The primary objective was to identify SD-RLKs and LRR-RLKs which demonstrated up-regulation in response to PAMPs. The SD-RLKs (At1g11330, At1g61430 and At1g61610) and LRR-RLKs (At1g51850, At2g19190 and At5g45840) were selected on the basis of microarray data (Nürnberger - TAIR accession set 100808727) and the Genevestigator database, and characterized utilizing bioinformatics tools. Here, molecular techniques were used to show that the selected RLK genes were responsive to PAMP inductions. Furthermore, this study explored which cis-elements and their corresponding transcription factors (TFs) are found in the promoter of plant defense genes and that may be involved in transcriptional regulation thereof...
- Full Text:
- Authors: New, Sherrie-Ann
- Date: 2013-07-29
- Subjects: Plant defenses , Plant-pathogen relationships , Arabidopsis thaliana - Disease and pest resistance
- Type: Thesis
- Identifier: uj:7708 , http://hdl.handle.net/10210/8573
- Description: M.Sc. (Biochemistry) , Our comprehension of pathogen perception and defense response mechanisms that play key roles in the resistance of plants against pathogen attack have progressed substantially within the recent years. Recognizing the molecular mechanisms involved in pathogen perception is the basis of understanding the signalling networks that are involved, including the transcriptional regulation of plant defense genes. This has proven to be a great challenge in plant pathology and, as such, has attracted much attention. The receptor-like kinases (RLKs) constitute one of the largest classes of plant defense genes in Arabidopsis thaliana, and contains, inter alia, the well-known leucine-rich repeats-RLKs (LRR-RLK), as well as the S-domain receptor-like kinases (SD-RLKs) that have been shown to be involved in pathogen perception and not only self-incompatibility (SI) as originally discovered. Some members of these RLKs are able to detect pattern-associated molecular patterns (PAMPs), which are conserved pathogen-derived molecules, and trigger a battery of basal defense responses. The transcriptional activation and expression levels of RLKs are dependent on the variation in promoter architecture as a result of the number, location, order and class of cis-elements found in a promoter sequence. It is hypothesized that candidate RLK genes involved in PAMP surveillance are triggered and transcriptionally regulated in response to perception of PAMPs, and that the intensity of response is relative to the promoter architecture. The primary objective was to identify SD-RLKs and LRR-RLKs which demonstrated up-regulation in response to PAMPs. The SD-RLKs (At1g11330, At1g61430 and At1g61610) and LRR-RLKs (At1g51850, At2g19190 and At5g45840) were selected on the basis of microarray data (Nürnberger - TAIR accession set 100808727) and the Genevestigator database, and characterized utilizing bioinformatics tools. Here, molecular techniques were used to show that the selected RLK genes were responsive to PAMP inductions. Furthermore, this study explored which cis-elements and their corresponding transcription factors (TFs) are found in the promoter of plant defense genes and that may be involved in transcriptional regulation thereof...
- Full Text:
Differential gene expression in Nicotiana tabacum cells in response to isonitrosoacetophenone
- Authors: Maake, Mmapula Peggy
- Date: 2013-12-09
- Subjects: Gene expression , Nicotiana tabacum , Tobacco - Diseases and pest resistance , Plant immunology , Plant-pathogen relationships , Plant defenses
- Type: Thesis
- Identifier: uj:7850 , http://hdl.handle.net/10210/8744
- Description: M.Sc. (Biochemistry) , Plants respond to various stress stimuli by activating a broad-spectrum of defence responses that can be expressed locally at the site of pathogen infection (hypersensitive response-HR) as well as systemically in uninfected tissue (systemic acquired resistance-SAR). The ability to continuously respond to both abiotic and biotic stimuli leads to changes in the plants’ physiology, morphology and development. Therefore, there is a need to define and understand the mechanism of the plant defence system, including the mode of recognition, activation of signalling pathways and subsequent defence. In so doing, a long lasting and effective protection against various pathogens may be established. In the current study, the transcriptome status of cultured cells of Nicotiana tabacum was investigated using annealing control primer (ACP)-based differential display (DD) since it is an improved technology to compare patterns of gene expression in RNA samples, isolated from tissue / cells under different biological conditions, using a novel priming system. Here, ACP-DDRT-PCR was used in combination with a next-generation sequencing technology, namely 454 pyro-sequencing, which is the only technique that generates longer reads which are suitable for de novo assembly and annotation of non-model plants like tobacco of which the genome is not yet published in Genbank. SAR occurs following induction by biotrophic or necrotising pathogens. However, it can also be manifested artificially after chemical treatment. In this study, isonitrosoacetophenone (INAP), a novel compound that was originally isolated from extracts of citrus peel undergoing oxidative stress, was used as a chemical inducer and it was hypothesised that this compound induces defence-related responses in plants. In order to investigate this, tobacco cell suspensions were elicited with 1 mM INAP, followed by ACP-DDRT-PCR and subsequent identification of differentially expressed genes using pyro-sequencing.
- Full Text:
- Authors: Maake, Mmapula Peggy
- Date: 2013-12-09
- Subjects: Gene expression , Nicotiana tabacum , Tobacco - Diseases and pest resistance , Plant immunology , Plant-pathogen relationships , Plant defenses
- Type: Thesis
- Identifier: uj:7850 , http://hdl.handle.net/10210/8744
- Description: M.Sc. (Biochemistry) , Plants respond to various stress stimuli by activating a broad-spectrum of defence responses that can be expressed locally at the site of pathogen infection (hypersensitive response-HR) as well as systemically in uninfected tissue (systemic acquired resistance-SAR). The ability to continuously respond to both abiotic and biotic stimuli leads to changes in the plants’ physiology, morphology and development. Therefore, there is a need to define and understand the mechanism of the plant defence system, including the mode of recognition, activation of signalling pathways and subsequent defence. In so doing, a long lasting and effective protection against various pathogens may be established. In the current study, the transcriptome status of cultured cells of Nicotiana tabacum was investigated using annealing control primer (ACP)-based differential display (DD) since it is an improved technology to compare patterns of gene expression in RNA samples, isolated from tissue / cells under different biological conditions, using a novel priming system. Here, ACP-DDRT-PCR was used in combination with a next-generation sequencing technology, namely 454 pyro-sequencing, which is the only technique that generates longer reads which are suitable for de novo assembly and annotation of non-model plants like tobacco of which the genome is not yet published in Genbank. SAR occurs following induction by biotrophic or necrotising pathogens. However, it can also be manifested artificially after chemical treatment. In this study, isonitrosoacetophenone (INAP), a novel compound that was originally isolated from extracts of citrus peel undergoing oxidative stress, was used as a chemical inducer and it was hypothesised that this compound induces defence-related responses in plants. In order to investigate this, tobacco cell suspensions were elicited with 1 mM INAP, followed by ACP-DDRT-PCR and subsequent identification of differentially expressed genes using pyro-sequencing.
- Full Text:
Differential proteomic analysis of Lipopolysaccharide-responsive proteins in Nicotiana tabacum
- Authors: Gerber, Isak B.
- Date: 2008-05-22T07:35:16Z
- Subjects: Tobacco disease and pest resistance , Plant-pathogen relationships , Plant defenses , Plant proteins , Plant proteomics
- Type: Thesis
- Identifier: uj:2083 , http://hdl.handle.net/10210/442
- Description: Prof. I.A. Dubery
- Full Text:
- Authors: Gerber, Isak B.
- Date: 2008-05-22T07:35:16Z
- Subjects: Tobacco disease and pest resistance , Plant-pathogen relationships , Plant defenses , Plant proteins , Plant proteomics
- Type: Thesis
- Identifier: uj:2083 , http://hdl.handle.net/10210/442
- Description: Prof. I.A. Dubery
- Full Text:
Development of analytical procedures for the quantification and evaluation of the roles of plant hormones involved in defense and induced resistance in plants
- Authors: Riet, Katlego Botlhaleng
- Date: 2015
- Subjects: Plant hormones , Plant defenses , Plant-pathogen relationships
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/58825 , uj:16496
- Description: Abstract: Please refer to full text to view abstract , M.Sc. (Biochemistry)
- Full Text:
- Authors: Riet, Katlego Botlhaleng
- Date: 2015
- Subjects: Plant hormones , Plant defenses , Plant-pathogen relationships
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/58825 , uj:16496
- Description: Abstract: Please refer to full text to view abstract , M.Sc. (Biochemistry)
- Full Text:
Metabolite profiling of defence-related secondary metabolites in tobacco cells, in response to ergosterol, a steroid from fungal membranes
- Authors: Tugizimana, Fidele
- Date: 2012-11-05
- Subjects: Metabolites , Fungal metabolites , Plant defenses , Metabolitic profile tests , Tobacco plants
- Type: Thesis
- Identifier: uj:7339 , http://hdl.handle.net/10210/8088
- Description: M.Sc. , Plants have the ability to continuously respond to various stimuli which alter their physiology, morphology and development. These stimuli may be abiotic or biotic and range from essential to toxic in their effects. One of these stimuli is a steroid from fungal membranes, ergosterol (C28H44O), which does not occur in plants. Ergosterol acts as a pathogen-associated molecular pattern molecule and triggers defence mechanisms in plants, characterised by highly regulated and interrelated events that include the elicitation of the oxidative burst and expression of a number of defencerelated genes. However, the ergosterol-induced global cellular reprogramming of the host has not been fully investigated in all aspects. No metabolomic study has previously been conducted to elucidate, for instance, the effect of ergosterol on plant metabolism. A clear and broader understanding of the molecular mechanisms involved in plant : ergosterol interactions is of paramount importance, for it would open up possibilities of developing novel, more effective and sustainable strategies to control or eradicate fungal diseases in plants. In plants, the metabolome is a compilation of all primary and secondary metabolites. The latter are the final recipients of genetic information, and their levels can influence gene expression and protein stability. Metabolite patterns reveal the actual cellular dynamic environment. Hence, qualitative and quantitative measurements of extra- and intracellular metabolites yield insights into the cellular processes that control the biochemical phenotype of the cell, tissue or whole organism. Metabolomics, the most recent of the ‘omics’ approaches, is the holistic analysis of metabolites present within a biological system under specific physiological conditions. In the present study a metabolomic approach was used to elucidate and analyse changes in the metabolism of tobacco (Nicotiana tabacum) cells following ergosterol treatment. Special attention is given to sesquiterpenoids since the antimicrobial compounds (phytoalexins) isolated from plants within the Solanaceae are mostly bicyclic sesquiterpenoids. Suspension of tobacco cells were treated with different concentrations (0 - 1000 nM) of ergosterol and incubated for different time periods (0 - 24 h). A viability assay, based on the ability of viable cells to reduce 2,3,5- triphenyltetrazolium chloride (TTC), was used to determine whether cell death occurred due to ergosterol treatment. No loss of cell viability was observed over the concentration range and time periods used in this study, indicating that the observed responses were due to the treatment alone and possible secondary responses due to cell death could be excluded. Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro extraction and a general methanol extraction. Chromatographic techniques (TLC/HPTLC, GC-FID, GC-MS, GC×GC-TOF-MS, UPLC-MS) and 1H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract interpretable information from the multidimensional data generated from the aforementioned techniques.
- Full Text:
- Authors: Tugizimana, Fidele
- Date: 2012-11-05
- Subjects: Metabolites , Fungal metabolites , Plant defenses , Metabolitic profile tests , Tobacco plants
- Type: Thesis
- Identifier: uj:7339 , http://hdl.handle.net/10210/8088
- Description: M.Sc. , Plants have the ability to continuously respond to various stimuli which alter their physiology, morphology and development. These stimuli may be abiotic or biotic and range from essential to toxic in their effects. One of these stimuli is a steroid from fungal membranes, ergosterol (C28H44O), which does not occur in plants. Ergosterol acts as a pathogen-associated molecular pattern molecule and triggers defence mechanisms in plants, characterised by highly regulated and interrelated events that include the elicitation of the oxidative burst and expression of a number of defencerelated genes. However, the ergosterol-induced global cellular reprogramming of the host has not been fully investigated in all aspects. No metabolomic study has previously been conducted to elucidate, for instance, the effect of ergosterol on plant metabolism. A clear and broader understanding of the molecular mechanisms involved in plant : ergosterol interactions is of paramount importance, for it would open up possibilities of developing novel, more effective and sustainable strategies to control or eradicate fungal diseases in plants. In plants, the metabolome is a compilation of all primary and secondary metabolites. The latter are the final recipients of genetic information, and their levels can influence gene expression and protein stability. Metabolite patterns reveal the actual cellular dynamic environment. Hence, qualitative and quantitative measurements of extra- and intracellular metabolites yield insights into the cellular processes that control the biochemical phenotype of the cell, tissue or whole organism. Metabolomics, the most recent of the ‘omics’ approaches, is the holistic analysis of metabolites present within a biological system under specific physiological conditions. In the present study a metabolomic approach was used to elucidate and analyse changes in the metabolism of tobacco (Nicotiana tabacum) cells following ergosterol treatment. Special attention is given to sesquiterpenoids since the antimicrobial compounds (phytoalexins) isolated from plants within the Solanaceae are mostly bicyclic sesquiterpenoids. Suspension of tobacco cells were treated with different concentrations (0 - 1000 nM) of ergosterol and incubated for different time periods (0 - 24 h). A viability assay, based on the ability of viable cells to reduce 2,3,5- triphenyltetrazolium chloride (TTC), was used to determine whether cell death occurred due to ergosterol treatment. No loss of cell viability was observed over the concentration range and time periods used in this study, indicating that the observed responses were due to the treatment alone and possible secondary responses due to cell death could be excluded. Intracellular metabolites were extracted with two methods: a selective dispersive liquid-liquid micro extraction and a general methanol extraction. Chromatographic techniques (TLC/HPTLC, GC-FID, GC-MS, GC×GC-TOF-MS, UPLC-MS) and 1H NMR spectroscopy were used for quantitative and qualitative analyses. Multivariate data analyses (PCA and OPLS-DA models) were used to extract interpretable information from the multidimensional data generated from the aforementioned techniques.
- Full Text:
Metabolomic studies of biotransformation-related changes in plant metabolism in response to isonitrosoacetophenone treatment
- Authors: Madala, Ntakadzeni Edwin
- Date: 2013-07-24
- Subjects: Plants - Metabolism , Biotransformation (Metabolism) , Plant metabolites , Plant defenses
- Type: Thesis
- Identifier: uj:7667 , http://hdl.handle.net/10210/8535
- Description: D.Phil. (Biochemistry) , This thesis concerns a study of the effect of isonitrosoacetophenone on plant metabolism. Three different systems were investigated; cultured tobacco and sorghum cells as well as Arabidopsis thaliana plants, and a metabolomic approach was followed. Unlike most scientific studies, metabolomics is a discipline which is not driven by a specific hypothesis, but rather by the obtained data to add scientific insights to the topic under investigation. As such, the current study lacks a definite overarching hypothesis, but specific objectives were outlined and answered in each experimental chapter. This thesis is therefore presented as a compilation of nine chapters in which experimental/research work is described in Chapter 3- 8. It is important to note that each chapter is presented in accordance with the guidelines for the respective journal in which the corresponding manuscript was published or submitted to.
- Full Text:
- Authors: Madala, Ntakadzeni Edwin
- Date: 2013-07-24
- Subjects: Plants - Metabolism , Biotransformation (Metabolism) , Plant metabolites , Plant defenses
- Type: Thesis
- Identifier: uj:7667 , http://hdl.handle.net/10210/8535
- Description: D.Phil. (Biochemistry) , This thesis concerns a study of the effect of isonitrosoacetophenone on plant metabolism. Three different systems were investigated; cultured tobacco and sorghum cells as well as Arabidopsis thaliana plants, and a metabolomic approach was followed. Unlike most scientific studies, metabolomics is a discipline which is not driven by a specific hypothesis, but rather by the obtained data to add scientific insights to the topic under investigation. As such, the current study lacks a definite overarching hypothesis, but specific objectives were outlined and answered in each experimental chapter. This thesis is therefore presented as a compilation of nine chapters in which experimental/research work is described in Chapter 3- 8. It is important to note that each chapter is presented in accordance with the guidelines for the respective journal in which the corresponding manuscript was published or submitted to.
- Full Text:
Comparison between conventional and quantum dot labeling strategies for LPS binding studies in Arabidopsis thaliana
- Authors: Mgcina, Londiwe Siphephise
- Date: 2013-12-09
- Subjects: Arabidopsis thaliana - Disease and pest resistance , Plant-pathogen relationships , Plant defenses , Plant protoplasts , Binding sites (Biochemistry) , Gram-negative bacteria , Quantum dots
- Type: Thesis
- Identifier: uj:7844 , http://hdl.handle.net/10210/8739
- Description: M.Sc. (Biochemistry) , Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and is composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides. When LPS is recognized as a microbe-/pathogen-associated molecular pattern (M/PAMP), it not only induces an innate immune response in plants but also stimulates the development of defence responses such as the immediate release of reactive oxygen species/intermediates (ROS/I), pathogen-related (PR) gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterisation of the elusive LPS receptor/receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease crop plant losses due to pathogen attack. In mammals, LPS binds to a LPS binding protein (LBP) to form a LPS-LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defence response through the signalling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is, however, unknown in plants. To address the LPS perception mechanism in plants, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted in Arabidopsis thaliana protoplasts using LPS labeled with Alexa 488 hydrazide. Quantum dots (Qdots), which allow non-covalent hydrophobic labeling of LPS, were further also employed in similar Arabidopsis protoplast binding studies. These studies were conducted by fluorescence determination through the use of a BD FACS Aria flow cytometer. Although Alexa-labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems, and hence Qdots were included to address this question. The conjugation of Qdots to LPS was confirmed by transmission electron microscopy (TEM) and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the perception process is also reported using Wortmannin and Brefeldin A as suitable endo- and exocytosis inhibitors. Affinity, specificity and saturability as well as the role of endo- and exocytosis inhibition in LPS binding to protoplasts was ultimately demonstrated by both fluorophores, with the use of Qdots as a label proving to be a more sensitive strategy in comparison to the conventional Alexa 488 hydrazide label.
- Full Text:
- Authors: Mgcina, Londiwe Siphephise
- Date: 2013-12-09
- Subjects: Arabidopsis thaliana - Disease and pest resistance , Plant-pathogen relationships , Plant defenses , Plant protoplasts , Binding sites (Biochemistry) , Gram-negative bacteria , Quantum dots
- Type: Thesis
- Identifier: uj:7844 , http://hdl.handle.net/10210/8739
- Description: M.Sc. (Biochemistry) , Lipopolysaccharide (LPS) is a complex lipoglycan that is found in the outer membrane of Gram-negative bacteria and is composed of three regions namely the fatty acid Lipid A, a core region of short oligosaccharide chains and an O-antigen region of polysaccharides. When LPS is recognized as a microbe-/pathogen-associated molecular pattern (M/PAMP), it not only induces an innate immune response in plants but also stimulates the development of defence responses such as the immediate release of reactive oxygen species/intermediates (ROS/I), pathogen-related (PR) gene expression and activation of the hypersensitive response (HR), resulting in stronger subsequent pathogen interactions. The identification and characterisation of the elusive LPS receptor/receptor complex in plants is thus of importance, since understanding the mechanism of perception and specific signal transduction pathways will clarify, and lead to the advancement of, basal resistance in plants in order to decrease crop plant losses due to pathogen attack. In mammals, LPS binds to a LPS binding protein (LBP) to form a LPS-LBP complex which is translocated to myeloid differentiation 2 (MD2) with the presence/absence of its co-receptor, a glycosylphosphatidylinositol (GPI)-linked protein, CD14. The interaction occurs on the host membrane and triggers an inflammatory defence response through the signalling cascade activated by the interaction with Toll-like receptor 4 (TLR4). A similar LPS-receptor interaction is, however, unknown in plants. To address the LPS perception mechanism in plants, biological binding studies with regard to concentration, incubation time and temperature, affinity, specificity and saturation were conducted in Arabidopsis thaliana protoplasts using LPS labeled with Alexa 488 hydrazide. Quantum dots (Qdots), which allow non-covalent hydrophobic labeling of LPS, were further also employed in similar Arabidopsis protoplast binding studies. These studies were conducted by fluorescence determination through the use of a BD FACS Aria flow cytometer. Although Alexa-labeling does not affect the biological activity in mammalian studies, the same cannot necessarily be said for plant systems, and hence Qdots were included to address this question. The conjugation of Qdots to LPS was confirmed by transmission electron microscopy (TEM) and results illustrated higher fluorescence values as compared to Alexa-LPS fluorescence analysis. Furthermore, inhibition of the perception process is also reported using Wortmannin and Brefeldin A as suitable endo- and exocytosis inhibitors. Affinity, specificity and saturability as well as the role of endo- and exocytosis inhibition in LPS binding to protoplasts was ultimately demonstrated by both fluorophores, with the use of Qdots as a label proving to be a more sensitive strategy in comparison to the conventional Alexa 488 hydrazide label.
- Full Text:
Metabolomic studies of induced defense-related changes in Sorghum bicolor in response to the pathogen Colletotrichum sublineolum
- Authors: Tugizimana, Fidele
- Date: 2017
- Subjects: Metabolic profile tests , Metabolites , Plants - Metabolism , Plant defenses , Sorghum , Colletotrichum sublineolum
- Language: English
- Type: Doctoral (Thesis)
- Identifier: http://hdl.handle.net/10210/234010 , uj:23906
- Description: Ph.D. (Biochemistry) , Abstract: The complementary roles of hypothesis- and data-driven (deductive and inductive) science in the post-genomic era have become a reality, with a resurgence of interest in metabolism and the explosion of systems biology methodologies. This has led to a paradigm shift on how biology research is being conducted; and the aspiration and possibilities to understand the nature of life at the molecular level have never looked better (Kell & Oliver, 2004; Strange, 2005; Ray, 2010). In the post-genomic era, biological science has thus become increasingly more data-intensive (big-data) science, driving the “cycle of knowledge” via a data-driven (inductive) reasoning to generate new hypotheses and insights. The systems biology approaches – omics layers: genomics, transcriptomics, proteomics and metabolomics – have become the essential strategies driving the search for fully describing and understanding the complex and dynamic metabolism of a biological system in toto (Goodacre, 2005). Metabolism, being a cornerstone of life – with its intertwined network of enzyme-catalysed biochemical reactions – is also a major source of cellular information that integrates environmental cues with intracellular signals to coordinate decisions in processes such as nutrient utilisation, signalling or differentiation. Furthermore, the regulation of metabolism is highly complex and dynamic as it entails several interconnected layers of translational and transcriptional mechanisms to eventually affect enzyme thermodynamics and kinetics (Ray, 2010; Fuhrer & Zamboni, 2015). This reawakening of interest in metabolism stems from acknowledging the etiolation of the field by the cloud of molecular biology (predominated by the philosophical preference of deductive reasoning), and the realisation of the impossibility to address and answer numerous biological questions without tapping into the dynamics of metabolism. Recognising the reciprocal regulation of metabolism and other cellular processes – investigated through systems biology strategies – has revolutionised and is advancing our understanding of complex physiology, aiming at a comprehensive representation of biological systems in their respective ever-changing environments (Vidal, 2009; McKnight, 2010; Ray, 2010; Nanda et al., 2011; Sévin et al., 2015)...
- Full Text:
- Authors: Tugizimana, Fidele
- Date: 2017
- Subjects: Metabolic profile tests , Metabolites , Plants - Metabolism , Plant defenses , Sorghum , Colletotrichum sublineolum
- Language: English
- Type: Doctoral (Thesis)
- Identifier: http://hdl.handle.net/10210/234010 , uj:23906
- Description: Ph.D. (Biochemistry) , Abstract: The complementary roles of hypothesis- and data-driven (deductive and inductive) science in the post-genomic era have become a reality, with a resurgence of interest in metabolism and the explosion of systems biology methodologies. This has led to a paradigm shift on how biology research is being conducted; and the aspiration and possibilities to understand the nature of life at the molecular level have never looked better (Kell & Oliver, 2004; Strange, 2005; Ray, 2010). In the post-genomic era, biological science has thus become increasingly more data-intensive (big-data) science, driving the “cycle of knowledge” via a data-driven (inductive) reasoning to generate new hypotheses and insights. The systems biology approaches – omics layers: genomics, transcriptomics, proteomics and metabolomics – have become the essential strategies driving the search for fully describing and understanding the complex and dynamic metabolism of a biological system in toto (Goodacre, 2005). Metabolism, being a cornerstone of life – with its intertwined network of enzyme-catalysed biochemical reactions – is also a major source of cellular information that integrates environmental cues with intracellular signals to coordinate decisions in processes such as nutrient utilisation, signalling or differentiation. Furthermore, the regulation of metabolism is highly complex and dynamic as it entails several interconnected layers of translational and transcriptional mechanisms to eventually affect enzyme thermodynamics and kinetics (Ray, 2010; Fuhrer & Zamboni, 2015). This reawakening of interest in metabolism stems from acknowledging the etiolation of the field by the cloud of molecular biology (predominated by the philosophical preference of deductive reasoning), and the realisation of the impossibility to address and answer numerous biological questions without tapping into the dynamics of metabolism. Recognising the reciprocal regulation of metabolism and other cellular processes – investigated through systems biology strategies – has revolutionised and is advancing our understanding of complex physiology, aiming at a comprehensive representation of biological systems in their respective ever-changing environments (Vidal, 2009; McKnight, 2010; Ray, 2010; Nanda et al., 2011; Sévin et al., 2015)...
- Full Text:
Stress protein expression and cell survival in tomato in response to Ralstonia solanacearum exposure
- Authors: Byth, Heather-Anne
- Date: 2012-08-20
- Subjects: Ralstonia solanacearum , Plant-pathogen relationships , Plant defenses , Heat shock proteins , Tomato wilts
- Type: Thesis
- Identifier: uj:2813 , http://hdl.handle.net/10210/6250
- Description: M.Sc. , Plants are in constant conflict with pathogens and have evolved intricate mechanisms to protect themselves against pathogens. The gene-for-gene response is regarded as the first line of defence when plant and pathogen meet. This interaction leads to the induction of defence proteins such as PR proteins that protect the plant from invading pathogens. A seemingly unrelated topic to plants and pathogens is heat shock proteins (HSP). HSP are a highly conserved group of defence proteins induced in all organisms in response to a variety of environmental stresses to provide protection from, and adaptation to cellular stress. HSP are in general not considered to be part of the defence response classically induced by avirulent pathogens and whether they are induced and play a role in plant-pathogen interactions is controversial. The protective chaperoning capacity of HSP makes them ideal proteins to exploit to target as endogenous defence proteins in the search for new strategies in the management of infectious diseases. In humans, HSP induction during infection is a complex phenomenon depending on the pathogen, whether the infection is acute or chronic, the host cell type and its differentiative state as well as environmental factors. In this investigation the expression of the inducible and constitutive isoforms of the 70kDa HSP (Hsp70/Hsc70) was investigated in tomato, Lycopersicon esculentum in response to virulent and avirulent strains of Ralstonia solanacearum, the causative agent of bacterial wilt. Expression of Hsp70 was studied in conjunction with the accumulation of PR-la and host cell viability. A quick, non-toxic, tetrazolium-based assay was developed from the Alamar Blue assay, commonly used in mammalian cells, and applied for the evaluation of host cell viability. The results shown suggest Hsp70/Hsc70 is significantly induced in tomato cell suspensions during an incompatible interaction 24h to 48 h following co-cultivation with the avirulent R. solanacearum strain compared to normal levels at this interval in cells exposed to the virulent strain. In both compatible and incompatible interactions Hsp70/Hsc70 levels eventually (72 h) accumulated correlating significantly with decreased viability. PR-la accumulation was significantly induced from 6 h to 18 h by the virulent as well as the avirulent R. solanacearum strains. In general, comparable results were obtained using leaf discs as an in vivo model. Based upon the differential induction of Hsp70/Hsc70 by virulent and avirulent pathogens it is proposed that HSP may play an important role in determining the outcome of the interaction between tomato and R. solanacearum. Successful defence may not only involve a limited number of defence genes but may result from a concerted action of a large number of defence genes.
- Full Text:
Stress protein expression and cell survival in tomato in response to Ralstonia solanacearum exposure
- Authors: Byth, Heather-Anne
- Date: 2012-08-20
- Subjects: Ralstonia solanacearum , Plant-pathogen relationships , Plant defenses , Heat shock proteins , Tomato wilts
- Type: Thesis
- Identifier: uj:2813 , http://hdl.handle.net/10210/6250
- Description: M.Sc. , Plants are in constant conflict with pathogens and have evolved intricate mechanisms to protect themselves against pathogens. The gene-for-gene response is regarded as the first line of defence when plant and pathogen meet. This interaction leads to the induction of defence proteins such as PR proteins that protect the plant from invading pathogens. A seemingly unrelated topic to plants and pathogens is heat shock proteins (HSP). HSP are a highly conserved group of defence proteins induced in all organisms in response to a variety of environmental stresses to provide protection from, and adaptation to cellular stress. HSP are in general not considered to be part of the defence response classically induced by avirulent pathogens and whether they are induced and play a role in plant-pathogen interactions is controversial. The protective chaperoning capacity of HSP makes them ideal proteins to exploit to target as endogenous defence proteins in the search for new strategies in the management of infectious diseases. In humans, HSP induction during infection is a complex phenomenon depending on the pathogen, whether the infection is acute or chronic, the host cell type and its differentiative state as well as environmental factors. In this investigation the expression of the inducible and constitutive isoforms of the 70kDa HSP (Hsp70/Hsc70) was investigated in tomato, Lycopersicon esculentum in response to virulent and avirulent strains of Ralstonia solanacearum, the causative agent of bacterial wilt. Expression of Hsp70 was studied in conjunction with the accumulation of PR-la and host cell viability. A quick, non-toxic, tetrazolium-based assay was developed from the Alamar Blue assay, commonly used in mammalian cells, and applied for the evaluation of host cell viability. The results shown suggest Hsp70/Hsc70 is significantly induced in tomato cell suspensions during an incompatible interaction 24h to 48 h following co-cultivation with the avirulent R. solanacearum strain compared to normal levels at this interval in cells exposed to the virulent strain. In both compatible and incompatible interactions Hsp70/Hsc70 levels eventually (72 h) accumulated correlating significantly with decreased viability. PR-la accumulation was significantly induced from 6 h to 18 h by the virulent as well as the avirulent R. solanacearum strains. In general, comparable results were obtained using leaf discs as an in vivo model. Based upon the differential induction of Hsp70/Hsc70 by virulent and avirulent pathogens it is proposed that HSP may play an important role in determining the outcome of the interaction between tomato and R. solanacearum. Successful defence may not only involve a limited number of defence genes but may result from a concerted action of a large number of defence genes.
- Full Text:
Identification of lipopolysaccharide-interacting plasma membrane proteins in Arabidopsis thaliana
- Authors: Vilakazi, Cornelius Sipho
- Date: 2015-11-12
- Subjects: Endotoxins , Arabidopsis thaliana - Defenses , Plant defenses , Plant gene expression
- Type: Thesis
- Identifier: uj:14554 , http://hdl.handle.net/10210/15087
- Description: M.Sc. (Biochemistry) , During microbial invasion, a variety of defense responses are induced in host plants. In order for host plants to combat potential diseases induced by microbes, they must be equipped with pattern recognition receptors (PRRs) localized at the cell surface, since such receptors enable the perception of conserved microbial epitopes termed microbe/pathogen-associated molecular patterns (M/PAMPs), thereby resulting in the activation of plant innate immunity via M/PAMP-triggered immunity (P/MTI). Lipopolysaccharide (LPS) is the major component of the outer leaflet of the external membrane of Gram-negative bacteria. This thermo-stable lipoglycan is exposed towards the external environment and plays an important role in bacterial adaptation to external surroundings. LPS is recognized as a major M/PAMP in plants, and thus potentiates or elicits defense-related responses such as the production of antimicrobial compounds and the expression of immune response genes. One of the most widely investigated effects of LPS on plants is its ability to prevent and/or suppress the hypersensitive response (HR) induced by an array of bacteria. The HR is a programmed cell death response which ends in a local necrosis of plant tissue, thereby resulting in a reduced number of viable bacteria that can further promote disease progression in the host.
- Full Text:
- Authors: Vilakazi, Cornelius Sipho
- Date: 2015-11-12
- Subjects: Endotoxins , Arabidopsis thaliana - Defenses , Plant defenses , Plant gene expression
- Type: Thesis
- Identifier: uj:14554 , http://hdl.handle.net/10210/15087
- Description: M.Sc. (Biochemistry) , During microbial invasion, a variety of defense responses are induced in host plants. In order for host plants to combat potential diseases induced by microbes, they must be equipped with pattern recognition receptors (PRRs) localized at the cell surface, since such receptors enable the perception of conserved microbial epitopes termed microbe/pathogen-associated molecular patterns (M/PAMPs), thereby resulting in the activation of plant innate immunity via M/PAMP-triggered immunity (P/MTI). Lipopolysaccharide (LPS) is the major component of the outer leaflet of the external membrane of Gram-negative bacteria. This thermo-stable lipoglycan is exposed towards the external environment and plays an important role in bacterial adaptation to external surroundings. LPS is recognized as a major M/PAMP in plants, and thus potentiates or elicits defense-related responses such as the production of antimicrobial compounds and the expression of immune response genes. One of the most widely investigated effects of LPS on plants is its ability to prevent and/or suppress the hypersensitive response (HR) induced by an array of bacteria. The HR is a programmed cell death response which ends in a local necrosis of plant tissue, thereby resulting in a reduced number of viable bacteria that can further promote disease progression in the host.
- Full Text:
Molecular characterisation of a lipopolysaccharide-induced S-domain receptor-like kinase from Nicotiana tabacum
- Authors: Sanabria, Natasha Mary-Anne
- Date: 2011-06-22T10:47:44Z
- Subjects: Plant-microbe relationships , Plants disease and pest resistance , Plant defenses , Gene expression , Endotoxins , Tobacco
- Type: Thesis
- Identifier: uj:7121 , http://hdl.handle.net/10210/3714
- Description: Ph.D. , Current models regarding plant : pathogen interactions assume that recognition of pathogen-associated molecular pattern (PAMP) molecules can occur through pattern recognition receptors (PRRs) on the surface of plant cells. Lipopolysaccharides (LPS) embedded in the cell wall of Gram-negative bacteria can trigger defence responses or prime the plant in order to respond more rapidly, following perception of bacterial pathogens. Limited data has been reported on signal transduction and the nature of the LPS receptors in plants since no receptors have been identified yet. Parallels have been shown to exist between self-incompatibility and pathogen recognition with regard to self / non-self recognition. The two processes were reviewed and conceptual and mechanistic links between microbial recognition and self-incompatibility were discussed herein. The role of S-domain receptor-like kinases (RLKs) in defence mechanisms has previously not been widely recognized or explored. It was reasoned that S-domain RLKs could be utilized to function as resistance (R) genes or as pattern recognition receptors in perception of PAMPs of a non-protein nature. It has been found that genes encoding receptors may be up-regulated in response to perception of its ligand. A putative receptor-like kinase was previously reported to be induced by LPS. This 153 bp differentially expressed transcript, HAP3-15 (GenBank accession number DR109311), might be an expressed sequence tag (EST) for a gene encoding a receptor for LPS. The experimental characterisation of this EST was reported herein. Gene-walking, reverse transcriptase polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), cloning, sequencing and bio-informatic analyses were used to identify the full gene. These results revealed that it encoded a receptor-like protein kinase with an extracellular S-domain recognition motif. The 2842 bp genomic sequence obtained, showed that the sequence had a defined promoter region and six major domains. The first five domains were encoded by the first exon. These domains included a B-lectin / agglutinin domain, an S-locus glycoprotein domain, an EGF-like repeat, a PAN domain, a transmembrane region and part of the 6th domain. The 6th domain was a kinase domain consisting of eleven sub-domains interspersed by three introns. The gene was therefore designated as the N. tabacum S-domain Receptor-like kinase (NS-RLK).
- Full Text:
- Authors: Sanabria, Natasha Mary-Anne
- Date: 2011-06-22T10:47:44Z
- Subjects: Plant-microbe relationships , Plants disease and pest resistance , Plant defenses , Gene expression , Endotoxins , Tobacco
- Type: Thesis
- Identifier: uj:7121 , http://hdl.handle.net/10210/3714
- Description: Ph.D. , Current models regarding plant : pathogen interactions assume that recognition of pathogen-associated molecular pattern (PAMP) molecules can occur through pattern recognition receptors (PRRs) on the surface of plant cells. Lipopolysaccharides (LPS) embedded in the cell wall of Gram-negative bacteria can trigger defence responses or prime the plant in order to respond more rapidly, following perception of bacterial pathogens. Limited data has been reported on signal transduction and the nature of the LPS receptors in plants since no receptors have been identified yet. Parallels have been shown to exist between self-incompatibility and pathogen recognition with regard to self / non-self recognition. The two processes were reviewed and conceptual and mechanistic links between microbial recognition and self-incompatibility were discussed herein. The role of S-domain receptor-like kinases (RLKs) in defence mechanisms has previously not been widely recognized or explored. It was reasoned that S-domain RLKs could be utilized to function as resistance (R) genes or as pattern recognition receptors in perception of PAMPs of a non-protein nature. It has been found that genes encoding receptors may be up-regulated in response to perception of its ligand. A putative receptor-like kinase was previously reported to be induced by LPS. This 153 bp differentially expressed transcript, HAP3-15 (GenBank accession number DR109311), might be an expressed sequence tag (EST) for a gene encoding a receptor for LPS. The experimental characterisation of this EST was reported herein. Gene-walking, reverse transcriptase polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE), cloning, sequencing and bio-informatic analyses were used to identify the full gene. These results revealed that it encoded a receptor-like protein kinase with an extracellular S-domain recognition motif. The 2842 bp genomic sequence obtained, showed that the sequence had a defined promoter region and six major domains. The first five domains were encoded by the first exon. These domains included a B-lectin / agglutinin domain, an S-locus glycoprotein domain, an EGF-like repeat, a PAN domain, a transmembrane region and part of the 6th domain. The 6th domain was a kinase domain consisting of eleven sub-domains interspersed by three introns. The gene was therefore designated as the N. tabacum S-domain Receptor-like kinase (NS-RLK).
- Full Text:
The effect of xenobiotics on the expression of a cytochrome p450 gene in Phaseolus vulgaris
- Authors: Basson, Adriaan Erasmus
- Date: 2012-08-08
- Subjects: Plant defenses , Cytochrome P-450 , Common bean , Plant-pathogen relationships , Phaseolus vulgaris
- Type: Thesis
- Identifier: uj:8976 , http://hdl.handle.net/10210/5446
- Description: M.Sc. , Plant cells have evolved the ability to detect pathogen ingress and subsequently activate defense-related functions as part of the plant pathogen response. One or more poorly defined signal transduction pathways, initiated upon recognition of the pathogen by the plant host, regulate expression of plant defense genes. Acquired resistance (AR) is an inducible defense mechanism exhibited by many plants that provides protection against a broad range of pathogens.Many chemical and environmental cues can elicit the same defenses or subsets therein. Cytochrome P450 enzymes are heme-dependent, mixed function oxidase systems that utilize dioxygen to produce a functionalized organic substrate and a molecule of water. They play important biosynthetic and detoxicative roles. They have been identified as being involved in the activation (e.g. allene oxide synthase) and execution of plant defense responses. To investigate the involvement of cytochromes P450 in the plant defense response - mainly through the activation of allene oxide synthases in the jamonate signaling pathway — Phaseolus vulgaris L.cv. Contender leaves were treated with chemical elicitors to mimic the plant-pathogen interaction and thereby activate plant defense responses. Through the use of differential display reverse transcription polymerase chain reaction and denaturing polyacrylamide gel electrophoresis, differentially expressed cDNA bands were isolated, cloned and sequenced. One of the cloned cDNA fragment proved to be a previously unreported cytochrome P450 cDNA, and was named CYP98A5. Dot blot analysis of bean leaves treated with various chemicals showed an enhanced expression of CYP98A5 in leaves treated with 3,5- dichlorosalicylic acid. Northern blot analysis of a time dependent induction study of CYP98A5 in bean leaves treated with this chemical compound indicated that 3,5-dichlorosalicylic acid induces CYP98A5 transcripts earlier than it is induced in control leaves. This might be indicative of a possible conditioning and sensitizing effect of 3,5- dichlorosalicylic acid on bean leaves to a more rapid and effective response with defense reactions once attacked by pathogens. CYP98A5, however, did not appear to be an allene oxide synthase when sequence comparison with other allene oxide synthases was performed; isolation and comparison of the complete CYP98A5 sequence could prove this wrong. It is not possible to assign any function to CYP98A5 at this stage; elucidation of the function of this enzyme in plants would provide more insight into this study.
- Full Text:
- Authors: Basson, Adriaan Erasmus
- Date: 2012-08-08
- Subjects: Plant defenses , Cytochrome P-450 , Common bean , Plant-pathogen relationships , Phaseolus vulgaris
- Type: Thesis
- Identifier: uj:8976 , http://hdl.handle.net/10210/5446
- Description: M.Sc. , Plant cells have evolved the ability to detect pathogen ingress and subsequently activate defense-related functions as part of the plant pathogen response. One or more poorly defined signal transduction pathways, initiated upon recognition of the pathogen by the plant host, regulate expression of plant defense genes. Acquired resistance (AR) is an inducible defense mechanism exhibited by many plants that provides protection against a broad range of pathogens.Many chemical and environmental cues can elicit the same defenses or subsets therein. Cytochrome P450 enzymes are heme-dependent, mixed function oxidase systems that utilize dioxygen to produce a functionalized organic substrate and a molecule of water. They play important biosynthetic and detoxicative roles. They have been identified as being involved in the activation (e.g. allene oxide synthase) and execution of plant defense responses. To investigate the involvement of cytochromes P450 in the plant defense response - mainly through the activation of allene oxide synthases in the jamonate signaling pathway — Phaseolus vulgaris L.cv. Contender leaves were treated with chemical elicitors to mimic the plant-pathogen interaction and thereby activate plant defense responses. Through the use of differential display reverse transcription polymerase chain reaction and denaturing polyacrylamide gel electrophoresis, differentially expressed cDNA bands were isolated, cloned and sequenced. One of the cloned cDNA fragment proved to be a previously unreported cytochrome P450 cDNA, and was named CYP98A5. Dot blot analysis of bean leaves treated with various chemicals showed an enhanced expression of CYP98A5 in leaves treated with 3,5- dichlorosalicylic acid. Northern blot analysis of a time dependent induction study of CYP98A5 in bean leaves treated with this chemical compound indicated that 3,5-dichlorosalicylic acid induces CYP98A5 transcripts earlier than it is induced in control leaves. This might be indicative of a possible conditioning and sensitizing effect of 3,5- dichlorosalicylic acid on bean leaves to a more rapid and effective response with defense reactions once attacked by pathogens. CYP98A5, however, did not appear to be an allene oxide synthase when sequence comparison with other allene oxide synthases was performed; isolation and comparison of the complete CYP98A5 sequence could prove this wrong. It is not possible to assign any function to CYP98A5 at this stage; elucidation of the function of this enzyme in plants would provide more insight into this study.
- Full Text:
Metabolite profiling of defence-related secondary metabolites in Solanum lycopersicum, in response to whitefly mediated begomovirus infection
- Authors: Rossouw, Leandri T.
- Date: 2016
- Subjects: Metabolic profile tests , Plants - Metabolism , Metabolites , Plant defenses
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/124936 , uj:20976
- Description: Abstract: Tomatoes (Solanum lycopersicum) are produced in all South African provinces and are considered to be the second most important (economically) and popular crop. South Africa produces an average of four hundred and fifty thousand (450 000) tons of tomatoes each year, grossing approximately R1,6 billion (ZAR) in 2011. The tomato industry supplies jobs to 22 500 people, with approximately 135 000 dependents. A large portion of these workers are minimum wage, low skilled labourers. Tomatoes are low in fat, free of cholesterol, a source of fibre, carbohydrates, vitamins C and A, potassium and antioxidants. Like all plants, tomato is subjected to attack by many potential pathogens, such as nematodes, bacteria, fungi and viruses. Viral diseases of tomato can have a large impact on tomato production and can cause large financial losses. Viruses of the genus Begomoviridae (family: Geminiviridea) constitute an important group of tomato pathogens and is currently the largest genus of all viral taxonomy (with 288 accepted species). Infection often causes devastating symptoms of leaf chlorosis, leaf cupping, stunted growth and reduced fruit set leading to large economic losses. Begomoviruses can be either monopartite or bipartite and are transmitted by the whitefly vector Bemisia tabaci. Whiteflies are polyphagous, plant-sucking insects with a very wide host range and as such are important invasive agricultural pests. Within the B. tabaci cryptic species complex, some members are extremely invasive, for instance Middle East - Asia minor 1 (MEAM1, also called B. argentifolii and biotype B) and Mediterranean (MED, also called biotype Q), are known worldwide as invasive. Some factors contributing to Begomoviridae currently being the largest viral taxonomy group includes the invasiveness of some B. tabaci species, as well as their ability to adapt to new host and new geographical regions. Another factor is the begomoviruses' well known propensity for recombination, which is the driving force of begomovirus evolution. In 1997 a new monopartite begomovirus was found to be infecting tomatoes in South Africa, causing devastating damage, that virus was named Tomato curly stunt virus (ToCSV). Although ToCSV currently remains the predominant begomovirus species infecting crops and weeds in tomato production areas in South Africa and Mozambique, many new species have been detected. In 2008 a new begomovirus, named Tomato curly stunt Mooketsi virus (ToCSMV), was detected in tomato plants in Mooketsi. In 2009 a second new begomovirus was detected in Noordoewer, it was named Tomato curly stunt Noordoewer virus (ToCSNV). And in 2010 another begomovirus was detected in Klawer, this viral isolate had previously been detected in Lanseria and thus was named Tomato curly stunt Lanseria virus (ToCSLV). In Chapter 2 these three new begomoviruses are characterized molecularly as well as phylogenetically. This chapter also describes the construction of infectious clones of these three viruses, which were used to study their symptom phenotype as well as fulfil Koch's... , M.Sc. (Biochemistry)
- Full Text:
- Authors: Rossouw, Leandri T.
- Date: 2016
- Subjects: Metabolic profile tests , Plants - Metabolism , Metabolites , Plant defenses
- Language: English
- Type: Masters (Thesis)
- Identifier: http://hdl.handle.net/10210/124936 , uj:20976
- Description: Abstract: Tomatoes (Solanum lycopersicum) are produced in all South African provinces and are considered to be the second most important (economically) and popular crop. South Africa produces an average of four hundred and fifty thousand (450 000) tons of tomatoes each year, grossing approximately R1,6 billion (ZAR) in 2011. The tomato industry supplies jobs to 22 500 people, with approximately 135 000 dependents. A large portion of these workers are minimum wage, low skilled labourers. Tomatoes are low in fat, free of cholesterol, a source of fibre, carbohydrates, vitamins C and A, potassium and antioxidants. Like all plants, tomato is subjected to attack by many potential pathogens, such as nematodes, bacteria, fungi and viruses. Viral diseases of tomato can have a large impact on tomato production and can cause large financial losses. Viruses of the genus Begomoviridae (family: Geminiviridea) constitute an important group of tomato pathogens and is currently the largest genus of all viral taxonomy (with 288 accepted species). Infection often causes devastating symptoms of leaf chlorosis, leaf cupping, stunted growth and reduced fruit set leading to large economic losses. Begomoviruses can be either monopartite or bipartite and are transmitted by the whitefly vector Bemisia tabaci. Whiteflies are polyphagous, plant-sucking insects with a very wide host range and as such are important invasive agricultural pests. Within the B. tabaci cryptic species complex, some members are extremely invasive, for instance Middle East - Asia minor 1 (MEAM1, also called B. argentifolii and biotype B) and Mediterranean (MED, also called biotype Q), are known worldwide as invasive. Some factors contributing to Begomoviridae currently being the largest viral taxonomy group includes the invasiveness of some B. tabaci species, as well as their ability to adapt to new host and new geographical regions. Another factor is the begomoviruses' well known propensity for recombination, which is the driving force of begomovirus evolution. In 1997 a new monopartite begomovirus was found to be infecting tomatoes in South Africa, causing devastating damage, that virus was named Tomato curly stunt virus (ToCSV). Although ToCSV currently remains the predominant begomovirus species infecting crops and weeds in tomato production areas in South Africa and Mozambique, many new species have been detected. In 2008 a new begomovirus, named Tomato curly stunt Mooketsi virus (ToCSMV), was detected in tomato plants in Mooketsi. In 2009 a second new begomovirus was detected in Noordoewer, it was named Tomato curly stunt Noordoewer virus (ToCSNV). And in 2010 another begomovirus was detected in Klawer, this viral isolate had previously been detected in Lanseria and thus was named Tomato curly stunt Lanseria virus (ToCSLV). In Chapter 2 these three new begomoviruses are characterized molecularly as well as phylogenetically. This chapter also describes the construction of infectious clones of these three viruses, which were used to study their symptom phenotype as well as fulfil Koch's... , M.Sc. (Biochemistry)
- Full Text:
Release of volatile compounds by Arabidopsis thaliana cells in response to elicitation by lipopolysaccharides
- Authors: Le Noury, Denise Anne
- Date: 2011-08-31T07:15:12Z
- Subjects: Arabidopsis thaliana , Plant gene expression , Plant defenses , Plant-pathogen relationships , Endotoxins , Lipopolysaccharides
- Type: Thesis
- Identifier: uj:7177 , http://hdl.handle.net/10210/3788
- Description: M.Sc. , Plants produce volatile organic compounds in response to certain elicitors and environments. These compounds have a variety of functions, including the attraction of insects for pollination and seed dispersal, responses to both abiotic and biotic stresses and the priming or sensitizing of neighbouring plants for subsequent attack. The majority of the volatile blend is made up of terpenoid compounds and these compounds are formed through the action of an important class of enzymes termed Terpene Synthases. Lipopolysaccharides form part of the cell surface of Gram-negative bacteria and they are classed as “pathogen-associated molecular pattern molecules” and are thought to induce defence responses in plants by influencing different metabolic pathways that could ultimately result in the production of defence volatiles. LPS from Burkholderia cepacia that has been reported to induce the oxidative burst, the nitric oxide burst and changes in cytosolic calcium concentrations, was used in this study. In order to analyse the volatiles, Single-Drop Microextraction and Solid-Phase Microextraction were used as static headspace sampling techniques that allow the preconcentration of volatile analytes prior to analysis. Both these techniques are fast, simple and equilibrium based and both allow for minimal sample size and preparation. Luminometry was performed in order to test the efficacy of LPS and to determine if LPS is able to induce the oxidative burst in Arabidopsis thaliana. Histochemical staining of transgenic plants containing the PR1:GUS and PDF:GUS reporter gene constructs was performed in order to determine which signalling pathway LPS follows, either the jasmonic acid pathway or the salicylic acid pathway. SPME was then used to extract samples from both time and concentration studies. The time studies involved incubation times of 0 h, 2 h, 4 h and 6 h and 0 d, 1 d, 2 d and 3 d respectively, while the concentration studies involved using LPS concentrations of 0, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml. SPME was also used for the comparision of two A. thaliana ecotypes (Columbia and C24) as well as two A. thaliana knock-out lines (At5g44630 – multi-product sesquiterpene synthase and At5g23960 – (E)-β-caryophyllene synthase), and finally it was used for the sampling of A. thaliana leaf tissue. SDME was used to compare two solvents, namely octane and toluene and these results were compared to the SPME results. GC-MS was used only for the identification of volatiles with both SPME and SDME. Finally, GC-MS was used with SPME to identify volatiles that are produced by leaf tissue after priming.
- Full Text:
- Authors: Le Noury, Denise Anne
- Date: 2011-08-31T07:15:12Z
- Subjects: Arabidopsis thaliana , Plant gene expression , Plant defenses , Plant-pathogen relationships , Endotoxins , Lipopolysaccharides
- Type: Thesis
- Identifier: uj:7177 , http://hdl.handle.net/10210/3788
- Description: M.Sc. , Plants produce volatile organic compounds in response to certain elicitors and environments. These compounds have a variety of functions, including the attraction of insects for pollination and seed dispersal, responses to both abiotic and biotic stresses and the priming or sensitizing of neighbouring plants for subsequent attack. The majority of the volatile blend is made up of terpenoid compounds and these compounds are formed through the action of an important class of enzymes termed Terpene Synthases. Lipopolysaccharides form part of the cell surface of Gram-negative bacteria and they are classed as “pathogen-associated molecular pattern molecules” and are thought to induce defence responses in plants by influencing different metabolic pathways that could ultimately result in the production of defence volatiles. LPS from Burkholderia cepacia that has been reported to induce the oxidative burst, the nitric oxide burst and changes in cytosolic calcium concentrations, was used in this study. In order to analyse the volatiles, Single-Drop Microextraction and Solid-Phase Microextraction were used as static headspace sampling techniques that allow the preconcentration of volatile analytes prior to analysis. Both these techniques are fast, simple and equilibrium based and both allow for minimal sample size and preparation. Luminometry was performed in order to test the efficacy of LPS and to determine if LPS is able to induce the oxidative burst in Arabidopsis thaliana. Histochemical staining of transgenic plants containing the PR1:GUS and PDF:GUS reporter gene constructs was performed in order to determine which signalling pathway LPS follows, either the jasmonic acid pathway or the salicylic acid pathway. SPME was then used to extract samples from both time and concentration studies. The time studies involved incubation times of 0 h, 2 h, 4 h and 6 h and 0 d, 1 d, 2 d and 3 d respectively, while the concentration studies involved using LPS concentrations of 0, 20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml and 100 μg/ml. SPME was also used for the comparision of two A. thaliana ecotypes (Columbia and C24) as well as two A. thaliana knock-out lines (At5g44630 – multi-product sesquiterpene synthase and At5g23960 – (E)-β-caryophyllene synthase), and finally it was used for the sampling of A. thaliana leaf tissue. SDME was used to compare two solvents, namely octane and toluene and these results were compared to the SPME results. GC-MS was used only for the identification of volatiles with both SPME and SDME. Finally, GC-MS was used with SPME to identify volatiles that are produced by leaf tissue after priming.
- Full Text:
Promoter analysis of members of a plant defense-related LRR-RLK gene cluster in Arabidopsis thaliana
- Authors: Mumm, Anina
- Date: 2014-07-15
- Subjects: Arabidopsis thaliana , Plant-pathogen relationships , Plant genome mapping , Plant defenses
- Type: Thesis
- Identifier: uj:11692 , http://hdl.handle.net/10210/11414
- Description: M.Sc. (Biochemistry) , A 14-member, closely-spaced cluster of genes coding for leucine-rich repeat receptor-like kinases (LRR-RLKs) is located on chromosome 1 of Arabidopsis thaliana. Following on from previous microarray studies that found some of the members of this cluster to be upregulated in response to biotic stressors, including the bacterial elicitor flg22, the present study sought to confirm, using a luciferase-based protoplast assay, that flg22 does in fact induce the expression of the genes, and then to investigate the promoters of the genes. The promoters of At1g51790, At1g51850 and At1g51890 responded positively in this particular assay, and bioinformatic analyses determined that W-boxes are over-represented in the cloned regions. Mutational inactivation of individual W-boxes in the promoter of At1g51790 drastically reduced the flg22 response, except for the W-box closest to the start site, which seemed to increase both basal and flg22-inducible expression. In the promoter of At1g51850, mutational inactivation of either or both of its W-box dyads resulted in virtually no flg22 inducibility. The deletion of 6 W-boxes in the promoter of At1g51890, done via truncation, drastically reduced both its basal expression and its inducible response to flg22. These results provide evidence that W-box cis-elements are responsible for the upregulation of these LRR-RLKs in response to flg22. WRKYs -7, -11, -22,and -26 were found bioinformatically to have similar expression patterns to some of the genes in the cluster, and are thus good candidates to investigate as transcriptional regulators of the cluster in future studies.
- Full Text:
Promoter analysis of members of a plant defense-related LRR-RLK gene cluster in Arabidopsis thaliana
- Authors: Mumm, Anina
- Date: 2014-07-15
- Subjects: Arabidopsis thaliana , Plant-pathogen relationships , Plant genome mapping , Plant defenses
- Type: Thesis
- Identifier: uj:11692 , http://hdl.handle.net/10210/11414
- Description: M.Sc. (Biochemistry) , A 14-member, closely-spaced cluster of genes coding for leucine-rich repeat receptor-like kinases (LRR-RLKs) is located on chromosome 1 of Arabidopsis thaliana. Following on from previous microarray studies that found some of the members of this cluster to be upregulated in response to biotic stressors, including the bacterial elicitor flg22, the present study sought to confirm, using a luciferase-based protoplast assay, that flg22 does in fact induce the expression of the genes, and then to investigate the promoters of the genes. The promoters of At1g51790, At1g51850 and At1g51890 responded positively in this particular assay, and bioinformatic analyses determined that W-boxes are over-represented in the cloned regions. Mutational inactivation of individual W-boxes in the promoter of At1g51790 drastically reduced the flg22 response, except for the W-box closest to the start site, which seemed to increase both basal and flg22-inducible expression. In the promoter of At1g51850, mutational inactivation of either or both of its W-box dyads resulted in virtually no flg22 inducibility. The deletion of 6 W-boxes in the promoter of At1g51890, done via truncation, drastically reduced both its basal expression and its inducible response to flg22. These results provide evidence that W-box cis-elements are responsible for the upregulation of these LRR-RLKs in response to flg22. WRKYs -7, -11, -22,and -26 were found bioinformatically to have similar expression patterns to some of the genes in the cluster, and are thus good candidates to investigate as transcriptional regulators of the cluster in future studies.
- Full Text:
Chemically induced defense responses in tobacco cell
- Authors: Louw, Anna Elizabeth
- Date: 2012-09-05
- Subjects: Plant defenses , Phytophthora nicotianae , Tobacco - Disease and pest resistance
- Type: Thesis
- Identifier: uj:3567 , http://hdl.handle.net/10210/6950
- Description: M.Sc. , Chemically-induced plant defense responses were investigated in tobacco cell cultures. The inducing conditions were as follows: chitosan (C), an elicitor (E) prepared from Phytophthora nicotianae, isonicotinic acid (INA), isonicotinamide (IND) and isonitrosoacetophenone (INAP) as well as the addition of INA, IND and INAP as conditioning agents (primary elicitors) followed by secondary elicitation with either chitosan or elicitor. The defense responses investigated included determinations of phenylalanine ammonia-lyase (PAL) activity, total soluble phenolic content, specific phenolic profiles, phytoalexin content, (3- 1,3-glucanase activity and electrophoretic analyses of pathogenesis-related proteins (PR). The compounds, 4-(3-methyl-2-butenoXy)isonitrosoacetophenone (0-INAP) and 2-isonitrosoacetophenone (INAP) were successfully synthesized from the starting materials p-hydroxyacetophenone and acetophenone respectively. The organic synthesis of 0-INAP involved the formation of a prenyl ether.of p-hydroxyacetophenone, followed by a nitrosation reaction using butyl nitrite as the source of the nitroso group, on the a-carbon atom adjacent to the carbonyl group. The synthesis of INAP only required a nitrosation reaction on the a-carbon atom adjacent to the carbonyl group. The yields of 0-INAP and INAP were 12 - 15 % and 80 %, respectively. An evaluation of the properties of 0-INAP indicated that the compound, dissolved in methanol, has a molar extinction coefficient of 16 5001.mor.cm - ' at A. 302 nm. The compound possesses antifungal activity against Cladosporium cucumerinum, Penicillium expansum and Aspergillus niger as well as the ability to scavenge superoxide radicals which was indicated by a decrease in the chemiluminescence signal produced in a reaction mbdure of hydrogen peroxide, horseradish peroxidase, the chemiluminescence probe, MCLA, and increasing concentrations of 0-INAP. The addition of INA to tobacco cells at a - final concentration of 12.5 iimol.g -1 cells or 2.5 mM did not lead to significant changes in PAL activity, but conditioning with INA, followed by chitosan as well as elicitor led to a 2.5-fold and a 4.3-fold induction respectively. INA as well as INA + C and INA + E led to significant increases in the total soluble phenolic content, and the HPLC analyses of these phenolics indicated the significant induction of a phenolic-like compound with a peak at Rt = 1.7 min. which possibly indicates isonicotinic acid, for INA + C and INA + E. A whole range of phytoalexins were detectable after the addition of INA to tobacco cells and conditioning with INA followed by chitosan induced the phytoalexin, lubimin, several hundred-fold. PR proteins were also induced by INA and a prominent band of 11- 13 kDa was induced after conditioning with INA, followed by secondary elicitation with the elicitor and especially with chitosan. (3-1,3-glucanase activity was also induced by INA; INA + E and particularly INA + C led to increases of 2.5-fold and 4.5-fold in 13-1,3-glucanase activity respectively. The addition of IND to tobacco cells at a final concentration of 12.5 pmol.g -1 cells or 2.5 mM led to a 2.6-fold induction in PAL activity after only 6 h, but conditioning with IND, followed by secondary elicitation did not lead to any significant changes. IND at the earlier time interval (24 h vs. 48 h) as well as IND + C and IND + E led to increases in the total soluble phenolic content, - and the HPLC analyses of these phenolics indicated the significant induction of a phenolic-like compound with ,a peak at Rt = 1.7 min. which possibly indicates isonicotinic acid, for IND + C and IND + E. A whole range of phytoalexins were detectable after the addition of IND to tobacco cells and conditioning with IND followed by chitosan induced the phytoalexin, solavetinone, several hundred-fold. PR proteins were also induced by IND and prominent bands of 34 kDa and 39 - 40 kDa were induced for IND + ELIC. (3-1,3-glucanase activity .was also induced by IND; however, secondary elicitation with chitosan did not lead to increases in enzyme activity, although a twofold increase was detectable for IND + ELIC, compared to IND 72. The addition of INAP to tobacco cells at a final concentration of 6.3 pmol.e cells or 1.25 mM led to a 1.7-fold induction in PAL activity after only 6 h, a response that was still detectable after 30 h; however, conditioning with INAP, followed by secondary elicitation did not lead to any noteworthy changes. INAP 24 as well as INAP 48 did not lead to significant changesin the total soluble phenolic content, but INAP + C and INAP+ E led to increases of 3.3-fold and 3.5-fold, respectively. HPLC analyses of the induced phenolics showed the significant induCtion of a phenolic compound with a peak at Rt = 14.5 min. which possibly indicate p-coumaric acid, for INAP + C and INAP + E. A whole range.of phytoalexins were detectable after the addition of INAP to tobacco cells, but the addition of a secondary elicitor led to a decrease in phytoalexin accumulation. PR proteins were also induced by INAP and conditioning with INAP, followed by especially the elicitor, led to the induction of a whole range of PR proteins with molecular masses ranging from 11 - 68 kDa. (3-1,3-glucanase activity was significantly induced (60-fold compared to control) by INAP 48; however, secondary elicitation led to a decrease in (3-1,3-glucanase)
- Full Text:
- Authors: Louw, Anna Elizabeth
- Date: 2012-09-05
- Subjects: Plant defenses , Phytophthora nicotianae , Tobacco - Disease and pest resistance
- Type: Thesis
- Identifier: uj:3567 , http://hdl.handle.net/10210/6950
- Description: M.Sc. , Chemically-induced plant defense responses were investigated in tobacco cell cultures. The inducing conditions were as follows: chitosan (C), an elicitor (E) prepared from Phytophthora nicotianae, isonicotinic acid (INA), isonicotinamide (IND) and isonitrosoacetophenone (INAP) as well as the addition of INA, IND and INAP as conditioning agents (primary elicitors) followed by secondary elicitation with either chitosan or elicitor. The defense responses investigated included determinations of phenylalanine ammonia-lyase (PAL) activity, total soluble phenolic content, specific phenolic profiles, phytoalexin content, (3- 1,3-glucanase activity and electrophoretic analyses of pathogenesis-related proteins (PR). The compounds, 4-(3-methyl-2-butenoXy)isonitrosoacetophenone (0-INAP) and 2-isonitrosoacetophenone (INAP) were successfully synthesized from the starting materials p-hydroxyacetophenone and acetophenone respectively. The organic synthesis of 0-INAP involved the formation of a prenyl ether.of p-hydroxyacetophenone, followed by a nitrosation reaction using butyl nitrite as the source of the nitroso group, on the a-carbon atom adjacent to the carbonyl group. The synthesis of INAP only required a nitrosation reaction on the a-carbon atom adjacent to the carbonyl group. The yields of 0-INAP and INAP were 12 - 15 % and 80 %, respectively. An evaluation of the properties of 0-INAP indicated that the compound, dissolved in methanol, has a molar extinction coefficient of 16 5001.mor.cm - ' at A. 302 nm. The compound possesses antifungal activity against Cladosporium cucumerinum, Penicillium expansum and Aspergillus niger as well as the ability to scavenge superoxide radicals which was indicated by a decrease in the chemiluminescence signal produced in a reaction mbdure of hydrogen peroxide, horseradish peroxidase, the chemiluminescence probe, MCLA, and increasing concentrations of 0-INAP. The addition of INA to tobacco cells at a - final concentration of 12.5 iimol.g -1 cells or 2.5 mM did not lead to significant changes in PAL activity, but conditioning with INA, followed by chitosan as well as elicitor led to a 2.5-fold and a 4.3-fold induction respectively. INA as well as INA + C and INA + E led to significant increases in the total soluble phenolic content, and the HPLC analyses of these phenolics indicated the significant induction of a phenolic-like compound with a peak at Rt = 1.7 min. which possibly indicates isonicotinic acid, for INA + C and INA + E. A whole range of phytoalexins were detectable after the addition of INA to tobacco cells and conditioning with INA followed by chitosan induced the phytoalexin, lubimin, several hundred-fold. PR proteins were also induced by INA and a prominent band of 11- 13 kDa was induced after conditioning with INA, followed by secondary elicitation with the elicitor and especially with chitosan. (3-1,3-glucanase activity was also induced by INA; INA + E and particularly INA + C led to increases of 2.5-fold and 4.5-fold in 13-1,3-glucanase activity respectively. The addition of IND to tobacco cells at a final concentration of 12.5 pmol.g -1 cells or 2.5 mM led to a 2.6-fold induction in PAL activity after only 6 h, but conditioning with IND, followed by secondary elicitation did not lead to any significant changes. IND at the earlier time interval (24 h vs. 48 h) as well as IND + C and IND + E led to increases in the total soluble phenolic content, - and the HPLC analyses of these phenolics indicated the significant induction of a phenolic-like compound with ,a peak at Rt = 1.7 min. which possibly indicates isonicotinic acid, for IND + C and IND + E. A whole range of phytoalexins were detectable after the addition of IND to tobacco cells and conditioning with IND followed by chitosan induced the phytoalexin, solavetinone, several hundred-fold. PR proteins were also induced by IND and prominent bands of 34 kDa and 39 - 40 kDa were induced for IND + ELIC. (3-1,3-glucanase activity .was also induced by IND; however, secondary elicitation with chitosan did not lead to increases in enzyme activity, although a twofold increase was detectable for IND + ELIC, compared to IND 72. The addition of INAP to tobacco cells at a final concentration of 6.3 pmol.e cells or 1.25 mM led to a 1.7-fold induction in PAL activity after only 6 h, a response that was still detectable after 30 h; however, conditioning with INAP, followed by secondary elicitation did not lead to any noteworthy changes. INAP 24 as well as INAP 48 did not lead to significant changesin the total soluble phenolic content, but INAP + C and INAP+ E led to increases of 3.3-fold and 3.5-fold, respectively. HPLC analyses of the induced phenolics showed the significant induCtion of a phenolic compound with a peak at Rt = 14.5 min. which possibly indicate p-coumaric acid, for INAP + C and INAP + E. A whole range.of phytoalexins were detectable after the addition of INAP to tobacco cells, but the addition of a secondary elicitor led to a decrease in phytoalexin accumulation. PR proteins were also induced by INAP and conditioning with INAP, followed by especially the elicitor, led to the induction of a whole range of PR proteins with molecular masses ranging from 11 - 68 kDa. (3-1,3-glucanase activity was significantly induced (60-fold compared to control) by INAP 48; however, secondary elicitation led to a decrease in (3-1,3-glucanase)
- Full Text:
Thermotolerance and Ralstonia solanacearum infection: implications for phenylpropanoid metabolism in Lycopersicon esculentum
- Authors: Kuun, Karolina
- Date: 2012-08-28
- Subjects: Plants, Effect of heat on , Plant defenses , Plant-pathogen relationships , Heat shock proteins , Plants - Metabolism , Ralstonia solanacearum
- Type: Thesis
- Identifier: uj:3372 , http://hdl.handle.net/10210/6771
- Description: M.Sc. , Field grown plants are constantly challenged with a variety of stressful factors, such as high temperatures, drought and pathogen infection that adversely affect crop production and quality. These stresses seldom occur as single entities in plants and in warm climates, heat stress is often a common dominator in combinatorial stress. The heat shock (HS) response in plants has priority over other stress responses, including the pathogen-induced stress response. Activation of the HS response prevents the normal plant defence strategy, leaving the plant vulnerable to pathogen attack. However, prior exposure to elevated temperatures confers protection from subsequent, otherwise lethal, temperatures (thermotolerance) and a variety of other stress conditions including heavy-metals, chilling injury and certain pathogens (cross tolerance). In general, litterature supports a central role for heat shock proteins (HSP), in particular the 70 kDa HSP (Hsp70), in thermotolerance. Incompatible host-pathogen interactions lead to the activation of an array of defence mechanisms, including the promotion of phenylpropanoid metabolism. Phenylalanine ammonia-lyase is a key regulator of this metabolic pathway, influencing the production of salicylic acid, lignin and phytoalexins among other essential defence products. In this study it was hypothesised that prior exposure to non-lethal HS confers protection from subsequent heat-related suppression of the phenylpropanoid pathway, induced as a defence mechanism during an incompatible plant-pathogen interaction. This hypothesis was verified by analysing the effect of thermotolerance on pathogen-related stimulation of PAL promoter activity, enzyme activity and lignin deposition. The tomato, Lycopersicon esculentum cultivar UC82B and Ralstonia solanacearum, the causative agent of bacterial wilt, were used as host-pathogen model. Specific objectives in the study were: (1) Development of PAL promoter-GUS reporter transformed Lycopersicon esculentum. (2) Establishment of a thermotolerance protocol that ensures optimal Hsp70 levels at subsequent HS. (3) Evaluation of the influence of prior heat treatment on phenylpropanoid metabolism after exposure to HS in combination with Ralstonia solanacearum. Results obtained support the hypothesis indicating that thermotolerance protects phenylpropanoid metabolism, in particular PAL promoter and enzyme activity, and to a certain extent lignin production, induced by avirulent Ralstonia solanacearum during a second severe HS. In contrast, HS without a prior heat treatment, suppressed phenylpropanoid metabolism. The protective potential of prior heat treatment during subsequent infection under hyperthermic conditions support the application of HSP in the development of novel plant protection strategies.
- Full Text:
- Authors: Kuun, Karolina
- Date: 2012-08-28
- Subjects: Plants, Effect of heat on , Plant defenses , Plant-pathogen relationships , Heat shock proteins , Plants - Metabolism , Ralstonia solanacearum
- Type: Thesis
- Identifier: uj:3372 , http://hdl.handle.net/10210/6771
- Description: M.Sc. , Field grown plants are constantly challenged with a variety of stressful factors, such as high temperatures, drought and pathogen infection that adversely affect crop production and quality. These stresses seldom occur as single entities in plants and in warm climates, heat stress is often a common dominator in combinatorial stress. The heat shock (HS) response in plants has priority over other stress responses, including the pathogen-induced stress response. Activation of the HS response prevents the normal plant defence strategy, leaving the plant vulnerable to pathogen attack. However, prior exposure to elevated temperatures confers protection from subsequent, otherwise lethal, temperatures (thermotolerance) and a variety of other stress conditions including heavy-metals, chilling injury and certain pathogens (cross tolerance). In general, litterature supports a central role for heat shock proteins (HSP), in particular the 70 kDa HSP (Hsp70), in thermotolerance. Incompatible host-pathogen interactions lead to the activation of an array of defence mechanisms, including the promotion of phenylpropanoid metabolism. Phenylalanine ammonia-lyase is a key regulator of this metabolic pathway, influencing the production of salicylic acid, lignin and phytoalexins among other essential defence products. In this study it was hypothesised that prior exposure to non-lethal HS confers protection from subsequent heat-related suppression of the phenylpropanoid pathway, induced as a defence mechanism during an incompatible plant-pathogen interaction. This hypothesis was verified by analysing the effect of thermotolerance on pathogen-related stimulation of PAL promoter activity, enzyme activity and lignin deposition. The tomato, Lycopersicon esculentum cultivar UC82B and Ralstonia solanacearum, the causative agent of bacterial wilt, were used as host-pathogen model. Specific objectives in the study were: (1) Development of PAL promoter-GUS reporter transformed Lycopersicon esculentum. (2) Establishment of a thermotolerance protocol that ensures optimal Hsp70 levels at subsequent HS. (3) Evaluation of the influence of prior heat treatment on phenylpropanoid metabolism after exposure to HS in combination with Ralstonia solanacearum. Results obtained support the hypothesis indicating that thermotolerance protects phenylpropanoid metabolism, in particular PAL promoter and enzyme activity, and to a certain extent lignin production, induced by avirulent Ralstonia solanacearum during a second severe HS. In contrast, HS without a prior heat treatment, suppressed phenylpropanoid metabolism. The protective potential of prior heat treatment during subsequent infection under hyperthermic conditions support the application of HSP in the development of novel plant protection strategies.
- Full Text:
- «
- ‹
- 1
- ›
- »