With the recent development of nanotechnology, there has been increased production of engineered nanomaterials but limited containment strategies, resulting in inevitable release of a large amount of engineered nanoparticles (ENPs) in the environment. Many ENPs have potential adverse impacts on the environment, and one of the most commonly used ENPs, silver nanoparticle (AgNP), has attracted increased global concern. The current study focused on phytotoxicity of AgNPs to a model plant, Arabidopsis thaliana. Silver nanoparticles were introduced into the growth medium for the wild type A. thaliana plants and root uptake and translocation of AgNPs were examined in hydroponic growth condition. We also conducted growth stage based phenotypic analysis by growing A. thaliana throughout its life cycle in soil. The result indicated that: (1) wild type seeds germination was not affected by either AgNPs or their dissolved Ag+ ions; (2) AgNPs exposure resulted in a concentration- and size- dependent inhibition effect to the root elongation; (3) confocal and electron microscopy indicated that AgNPs could be taken up by seedling roots, yet most of AgNPs attached to the surface of seedling root cap; (4) AgNPs and their dissolved Ag+ ions at tested concentrations had little influence on the vegetative growth of A. thaliana, but they accelerated the floral development; and (5) the effect on the floral development stage reduced the quality of second-generation (F1) seeds, as indicated by their lower germination rate. In conclusion, AgNPs displayed both acute and chronic phytotoxicity to A. thaliana.

Entre os minerais, o fósforo é um dos mais limitantes ao crescimento e desenvolvimento vegetal. Como é altamente requerido para os mais diversos processos fisiológicos e celulares, as plantas desenvolveram complexos mecanismos para manejar sua deficiência. As respostas à limitação de fósforo são bem conhecidas, mas sobre a sua percepção e a transdução do sinal pouco se sabe. Os mutantes p9, p23 e p37 estudados neste trabalho são provenientes de uma seleção que busca identificar genes regulatórios envolvidos na sinalização de fósforo. Desta forma, fez-se através da caracterização morfológica, fisiológica e bioquímica destes mutantes condicionais de Arabidopsis thaliana, deficientes quando ácidos nucléicos são a única fonte de fosfato (Pi), a ampliação do conhecimento da rota de sinalização da limitação de Pi. Os fenótipos dos mutantes devem-se à mutação em um gene recessivo para cada mutante, sendo estes complementares. Análises do sistema radicular, acúmulo de amido e antocianinas, teor de Pi livre e do P total e atividade de nucleases em diferentes disponibilidades de fósforo e a avaliação da especificidade dos fenótipos à deficiência de P possibilitaram a criação de hipóteses para a ação dos genes mutados A mutação de p9 causa, provavelmente, alterações na sensibidade às concentrações de Pi, podendo estar relacionada tanto aos sensores da raiz quanto à interação da transdução dos sinais entre o sensor local e o status da planta. Devido à limitação de sementes não foi possível desenvolver hipótese sobre a ação do gene mutado em p23. Enquanto o gene mutado em p37 age primordialmente sobre o elongamento e a divisão das células radiculares, estas respostas podem estar sendo influenciadas pelos níveis de citocinina. Assim, a complexidade da transdução do sinal à limitação de Pi e a interação com outras rotas de sinalização em plantas vasculares evidencia a importância de estudar suas respostas e esclarecer como esses processos são regulados.

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Entre os minerais, o fósforo é um dos mais limitantes ao crescimento e desenvolvimento vegetal. Como é altamente requerido para os mais diversos processos fisiológicos e celulares, as plantas desenvolveram complexos mecanismos para manejar sua deficiência. As respostas à limitação de fósforo são bem conhecidas, mas sobre a sua percepção e a transdução do sinal pouco se sabe. Os mutantes p9, p23 e p37 estudados neste trabalho são provenientes de uma seleção que busca identificar genes regulatórios envolvidos na sinalização de fósforo. Desta forma, fez-se através da caracterização morfológica, fisiológica e bioquímica destes mutantes condicionais de Arabidopsis thaliana, deficientes quando ácidos nucléicos são a única fonte de fosfato (Pi), a ampliação do conhecimento da rota de sinalização da limitação de Pi. Os fenótipos dos mutantes devem-se à mutação em um gene recessivo para cada mutante, sendo estes complementares. Análises do sistema radicular, acúmulo de amido e antocianinas, teor de Pi livre e do P total e atividade de nucleases em diferentes disponibilidades de fósforo e a avaliação da especificidade dos fenótipos à deficiência de P possibilitaram a criação de hipóteses para a ação dos genes mutados A mutação de p9 causa, provavelmente, alterações na sensibidade às concentrações de Pi, podendo estar relacionada tanto aos sensores da raiz quanto à interação da transdução dos sinais entre o sensor local e o status da planta. Devido à limitação de sementes não foi possível desenvolver hipótese sobre a ação do gene mutado em p23. Enquanto o gene mutado em p37 age primordialmente sobre o elongamento e a divisão das células radiculares, estas respostas podem estar sendo influenciadas pelos níveis de citocinina. Assim, a complexidade da transdução do sinal à limitação de Pi e a interação com outras rotas de sinalização em plantas vasculares evidencia a importância de estudar suas respostas e esclarecer como esses processos são regulados.

M.Sc. / On exposure to abiotic or biotic stresses, plants initiate a cascade of metabolic reactions, some of which lead to the biosynthesis of secondary metabolites with roles in self defense. Phytoalexins are a class of secondary metabolites synthesized de novo in response to microbial attack by activation of certain biosynthetic pathways. Cruciferae phytoalexins are all indole based with a carbon, nitrogen and sulfur containing constituent on the 3’ position of the indole ring. This common similarity of all Cruciferae phytoalexins suggests that the plants all share a common indole precursor. Camalexin is the primary phytoalexin of Arabidopsis thaliana. De novo synthesis of camalexin upon infection, as well as its antimicrobial nature supports its role in disease resistance. Evidence exists that suggests the inducible biosynthesis of camalexin involves steps of the tryptophan pathway, along with an increase in transcript and protein levels of the tryptophan pathway enzymes after microbial infection. Bacterial LPS (lipopolysaccharide) has been described as one of the microbe/pathogenassociated molecular patterns (M/PAMPs) capable of eliciting the activation of the plant innate immune system. LPS is an integral component of the cell surface of Gram-negative bacteria. It is a complex which is exposed to the external environment, and is thus involved with external interactions of the bacteria. The hypothesis investigated in this dissertation is that LPS, as a lipoglycan PAMP, results in activation of signal transduction pathways involved in defense that lead to the production of the defense metabolite, camalexin. Furthermore, that the genes CYP71B15, CYP79B2 and TSB are up-regulated in response to LPS during camalexin biosynthesis via the tryptophan pathway. To test this hypothesis, camalexin production was investigated through a combination of analytical techniques including thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC), ultra pressure liquid chromatography-mass spectrometry (UPLC-MS) and fluorescence spectroscopy. Genes in the camalexin biosynthetic pathway were investigated by two-step reverse transcription polymerase chain reaction (PCR), GUS reporter gene assays and quantitative real time PCR (RT-qPCR).

Ralstonia solanaearum, a soil borne pathogen infects several important crops causing wilting. In 2000-2001, two eucalyptus isolates, BCCF 401 and BCCF 402 were isolated from plantations in Kwa-Zulu Natal and the Democratic Republic of Congo, respectively. Arabidopsis has been recognised as a host for R. solanacearum and as such has been adopted as a model to understand the plant defence response against this pathogen. The aim of this study was to use microarray expression profiling techniques to elucidate the plant defence response and to identify candidate genes possibly contributing towards resistance against the pathogen. As a means to optimise microarray expression profiling, the differential expression in an Arabidopsis mutant, cir1 (constitutively induced resistance 1) and wild-type plants was investigated using a custom 500-probe microarray. Several genes were found to be induced in cir1 at a significance threshold of –log10(p) equal to 3 (p< 0.001) using a mixed model ANOVA approach. The genes AtACP1 (sodium inducible calcium binding protein), AtP2CHA (protein phosphatase 2C), AtGSTF7 (glutathione S transferase), tryptophan synthase betalike and AtPAL1 (phenylalanine ammonia lyase 1), AtEREBP-4 (ethylene response element binding protein 4) and HFR1 (long hypocotyl in far-red 1) were further identified as possible candidate genes which may contribute to disease resistance in cir1 against Pseudomonas syringae pv. tomato. A similar transcript profiling approach, using the optimised protocols, was adopted to investigate the compatible interaction between Arabidopsis ecotype Col-5 and the R. solanacearum isolate BCCF 401. A screen of 5000 Arabidopsis ESTs revealed approximately 120 genes differentially regulated by R. solanacearum infection at a significance threshold of p<0.03 (Bonferroni corrected). Subsequent bioinformatic comparisons revealed that abscisic acid responses appear to be induced in Col-5 in response to the pathogen and that R. solanacearum induces an expression profile consistent with a necrotroph. The basal defence responses in Col-5 against R. solanacearum infection were investigated by comparing the expression data to that during treatment with the pathogen associated molecular patterns (PAMPs) flg22 and lipopolysaccharide, and the Type Three Secretion System deficient Pst hrp- mutant. Expression patterns for a subset of these genes were suggestive of host basal defences manipulated by the pathogen. It is hypothesised that genetic engineering to alter the expression of these “pathogen-manipulated” genes could contribute to resistance against R. solanacearum in the host. Copyright 2008, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Naidoo, S 2008, Microarray expression studies in the model plant Arabidopsis thaliana infected with the bacterial pathogen Ralstonia solanacearum, PhD thesis, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-11182008-092625 / > D559/gm / Thesis (PhD)--University of Pretoria, 2008. / Plant Science / unrestricted

Intraspecific variation within the genetic model plant Arabidopsis thaliana has been used to research numerous potentially adaptive and economically important traits. In this thesis I used this tool to investigate root morphology and adaptation of plants to edaphic conditions. Firstly I tested local adaptation of chosen A. thaliana wild genotypes collected from north eastern Scotland, to two soil types with contrasting textures. When local adaptation is defined as fitness advantage (reproductive output) in local soil, I did not find clear signs of local adaptation. Additionally, I observed significant phenotypic variation between collected accessions for both mineral nutrient uptake and growth in the two soil types. Together these results may suggest the mixing of adapted genotypes due to extensive human disturbance in north eastern Scotland. Secondly I focused on two linked aspects of root biology: endodermal development and root system architecture. The characteristic features of the endodermis include Casparian strips (CS) that form a barrier to apoplastic transport. Some mutants with an altered CS are sensitive to growth on media with elevated magnesium (Mg) and reduced calcium (Ca). In order to potentially identify novel alleles of genes involved in CS biosynthesis I took advantage of this growth phenotype and performed genome wide association (GWA) analysis on response of plants to low Ca/Mg ratio of the growth medium. As a result I compiled a list of genes for the future research by choosing candidate genes identified in the GWA study for both plant weight and elemental composition. This list was further refined using knowledge on gene expression in the endodermis. Accessions with the most extreme response to this low Ca/Mg treatment were analysed further. I identify a link between both lateral root number and total root length with performance on growth medium containing a low Ca/Mg ratio.

Understanding the causal relationship between genotype and phenotype is a major objective in biology. The main interest is in understanding trait architecture and identifying loci contributing to the respective traits. Genome-wide association mapping (GWAS) is one tool to elucidate these relationships and has been successfully used in many different species. However, most studies concentrate on marginal marker effects and ignore epistatic and gene-environment interactions. These interactions are problematic to account for, but are likely to make major contributions to many phenotypes that are not regulated by independent genetic effects, but by more sophisticated gene-regulatory networks. Further complication arises from the fact that these networks vary in different natural accessions. However, understanding the differences of gene regulatory networks and gene-gene interactions is crucial to conceive trait architecture and predict phenotypes. The basic subject of this study – using data from the Arabidopsis 1001 Genomes Project – is the analysis of pre-mature stop codons. These have been incurred in nearly one-third of the ~ 30k genes. A gene-gene interaction network of the co-occurrence of stop codons has been built and the over and under representation of different pairs has been statistically analyzed. To further classify the significant over and under- represented gene-gene interactions in terms of molecular function of the encoded proteins, gene ontology terms (GO-SLIM) have been applied. Furthermore, co- expression analysis specifies gene clusters that co-occur over different genetic and phenotypic backgrounds. To link these patterns to evolutionary constrains, spatial location of the respective alleles have been analyzed as well. The latter shows clear patterns for certain gene pairs that indicate differential selection. / Das Verständnis des kausalen Zusammenhangs zwischen Genotyp und Phänotyp ist ein wichtiges Ziel in der Biologie. Das Hauptinteresse liegt darin, die Merkmalsarchitektur zu verstehen und Loci zu identifizieren, die zu den jeweiligen Merkmalen beitragen. Genome-wide association mapping (GWAS) ist ein Werkzeug, um diese Zusammenhänge aufzuklären und wurde erfolgreich in vielen verschiedenen Arten eingesetzt. Die meisten Studien konzentrieren sich jedoch auf marginale Markereffekte und ignorieren epistatische und Gen-Umwelt-Interaktionen. Diese Wechselwirkungen sind problematisch zu erklären, werden aber wahrscheinlich einen wichtigen Beitrag zu vielen Phänotypen leisten, die nicht durch unabhängige genetische Effekte, sondern durch ausgefeiltere genregulatorische Netzwerke reguliert werden. Eine weitere Komplikation ergibt sich aus der Tatsache, dass sich diese Netzwerke in verschiedenen natürlichen Akzessionen unterscheiden. Das Verständnis der Unterschiede zwischen genregulatorischen Netzwerken und Gen-Gen- Interaktionen ist jedoch entscheidend, um die Merkmalsarchitektur zu konzipieren und Phänotypen vorherzusagen. Das grundlegende Thema dieser Studie – unter Verwendung von Daten aus dem Arabidopsis 1001 Genomes Project – ist die Analyse von vorzeitigen Stop-Codons. Diese sind in fast einem Drittel der ~ 30k-Gene aufgetreten. Ein Gen-Gen- Interaktionsnetzwerk des gleichzeitigen Auftretens von Stop-Codons wurde aufgebaut und die Über- und Unterrepräsentation verschiedener Paare wurde statistisch analysiert. Um die signifikante über- und unterrepräsentierte Gen-Gen-Interaktion in Bezug auf den biologischen Prozess der kodierten Proteine weiter zu klassifizieren, wurden genonkologische Begriffe (GO-SLIM) verwendet. Darüber hinaus spezifiziert die Koexpressionsanalyse Gencluster, die über verschiedene genetische und phänotypische Hintergründe hinweg gleichzeitig auftreten. Um diese Muster mit evolutionären Einschränkungen in Verbindung zu bringen, wurde auch die räumliche Lage der jeweiligen Allele analysiert. Letzteres zeigt klare Muster für bestimmte Genepaare, die auf eine differentielle Selektion hinweisen.

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