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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

Plants have evolved sophisticated defence systems against pathogen infection. Initiation of induced defence signalling often involves specific recognition of invading pathogens by the products of specialized host Resistance (R) genes. Consequently, the pathogen is stopped at the site of infection. A unique dominant mutant in Arabidopsis thaliana, snc1, constitutively expresses pathogensis-related (PR) genes and exhibits enhanced resistance to bacterial and oomycete pathogens. SNC1 encodes an R-gene – a single amino acid change renders this protein constitutively active without interaction with pathogens. snc1 displays a stunted phenotype that may be caused by both the accumulation of toxic compounds and energy squandered on unnecessary defence instead of normal growth. The distinctive morphological phenotype of snc1 is intimately associated with the other resistance phenotypes, and provides a robust genetic tool for dissecting the signalling events downstream of snc1. To identify genes important for defence signalling, we carried out a suppressor screen to identify modifier of snc1 (mos) mutants that restore the wild type size and morphology in the snc1 background. Furthermore, in most cases, a loss of sneakiness in mos mutants correlated with a reduced or abolished constitutive PR gene expression, SA accumulation and pathogen resistance in snc1 plants. These loss of function mutants represent defects in positive regulators of the snc1 pathway. I cloned and characterized two mos mutants, and showed that they both have roles in Arabidopsis innate immunity as well. mos6 partially suppresses snc1 and exhibits enhanced disease susceptibility (EDS) to an oomycete pathogen. MOS6, identified by map-based cloning, encodes an alpha-importin subunit, one of 8 found in Arabidopsis, and has a demonstrated role in nucleocytoplasmic partitioning (protein import). Two other genes cloned by others from this screen, MOS3 and MOS7, encode components of the nuclear pore complex, implicating nuclear trafficking as a key regulator in plant innate immunity. mos4 exhibits EDS to virulent and avirulent bacterial and oomycete pathogens. There is evidence that MOS4-mediated resistance is independent of the signalling protein NPR1. MOS4 encodes a protein with homology to human Breast Cancer Amplified Sequence 2 and with predicted protein-protein interaction domains. Subcellular localization of MOS4-GFP shows that MOS4 is localized to the nucleus. To illuminate the biochemical function of MOS4, a yeast-2-hybrid screen was conducted. One MOS4-interactor was a putative myb transcription factor, MOS4-Associated Complex Protein 1 (MAC1), also known at AtCDC5. MAC1 interacts directly with MOS4 in vitro and in planta. mac1 insertional mutants exhibit defects in immune responses to pathogens similar to that of mos4. In addition, mac1 also partially suppressed snc1 morphology and enhanced resistance. Both MOS4 and MAC1 have homologs in humans and fission yeast that are members of a discrete protein complex that has been implicated in several different biological processes including RNA splicing, apoptosis and protein degradation. Using proteomics data from yeast and human, we found genes with homology to additional components of the orthologous complex in Arabidopsis, and isolated insertion mutants in these. Mutations in PRL1, which encodes a WD protein, display similar disease phenotypes to that of mos4 and mac1. AtCDC5 has DNA binding activity, suggesting that this complex may regulate defence responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be identified function in mammalian innate immunity. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

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.

In the model cruciferous plant, Arabidopsis thaliana, tryptophan (Trp) is a focal point for growth and defense as it is used for the production of secondary metabolites including the growth hormone indole-3-acetic acid (IAA, or auxin), and two classes of defense compounds: indole glucosinolates (IGs) and camalexin. Trp metabolism in plants is of general importance to agriculture because animals (including humans) cannot synthesize Trp and must obtain it from their diet. Questions remain about the synthesis and regulation of Trp and how it relates to secondary metabolism in Arabidopsis. In this thesis it is shown that IGs are a sink for Trp metabolism because auxotrophic mutants deficient in Trp production are suppressed in combination with the IG-deficient cyp79B2 cyp79B3 mutant and enhanced in combination with IG overproducing mutant, atr1D. Because Trp auxotrophic mutants were found to produce IGs, the four predicted Arabidopsis Trp Synthase Beta genes (TSB1, TSB2, TSB3 and TSBt2) were examined for their role in Trp primary and secondary metabolism. It was determined that members of this gene family, while being redundant for enzyme activity, may have unique functions in channeling Trp to different secondary endpoints. tsb1 tsb2 plants display a healthier phenotype and produce lower IG levels than the single tsb1 mutants, in contrast to tsb1 tsbt2 plants, which have elevated IG production and an enhanced auxotrophic phenotype. tsb2 tsbt2 plants are indiscernible from WT. Gene expression in Trp biosynthetic pathway steps, IG biosynthesis genes, and regulatory TFs is dysregulated in these mutants. In a second part of this thesis, transcriptional regulation of IG synthesis was examined with respect to tissue specificity and stress. In collaboration with Judith Bender's laboratory at Brown University, the function of a subfamily of three Myb transcription factors that have been implicated in regulating IG biosynthesis genes was studied. Using combinations of Myb knockout mutants and GUS reporter plants, tissue specific roles for MYB34 and MYB51 in root and shoot tissues, respectively, were found. In addition, roles were discovered for MYB34 in mediating anti-herbivory signals, and for both MYB51 and MYB122 in regulating defense against microbial pathogens.

Myzus persicae (Sülzer), commonly known as the green peach aphid (GPA), is a polyphagous insect that can infest over 100 families of economically important plants and is major pest for vegetable crops. This study utilizes the Arabidopsis-GPA model system with the aim to elucidate the role of the plant disaccharide trehalose in providing defense against GPA. This study demonstrates a novel role for TPS11 in providing defense against GPA. TPS11 expression was found to be transiently induced in Arabidopsis plants in response to GPA infestation and the TPS11 gene was required for curtailing GPA infestation. TPS11, which encodes for trehalose phosphate synthase and phosphatase activities, contributes to the transient increase in trehalose in the GPA infested tissues. This work suggests that TPS11-dependent trehalose has a signaling function in plant defense against GPA. in addition, trehalose also has a more direct role in curtailing GPA infestation on Arabidopsis. This work also shows that TPS11 is able to modulate both carbohydrate metabolism and plant defenses in response to GPA infestation. the expression of PAD4, an Arabidopsis gene required for phloem-based defenses against GPA, was found to be delayed in GPA infested tps11 mutant plants along with increased sucrose levels and lower starch levels as compared to the GPA infested wild type plants. This work provides clear evidence that starch metabolism in Arabidopsis is altered in response to GPA feeding and that TPS11-modulated increase in starch contributes to the curtailment of GPA infestation in Arabidopsis.

RNA decay plays an essential role in the regulation of gene expression during plant development and response to environmental stimuli. The protein DXO is a 5' to 3' exonuclease that functions in RNA degradation and RNA quality control that has been studied in animals. It has not yet been identified in plants. The gene locus At4g17620 in Arabidopsis thaliana encodes a protein homolog of the mammalian DXO, termed AtDXO. Recombinantly expressed AtDXO possesses a 5'-3' RNA exonuclease activity in vitro. Loss-of-function of AtDXO in Arabidopsis generates multiple growth defects, including curled and yellowish leaves, growth retardation and limited fertility, whereas overexpression show no obvious growth phenotype. The development defect of atdxo might be attributed to aberrant RNAs, which are not degraded when AtDXO is dysfunctioning. From the RNA-Seq analysis, the transcriptome pattern of atdxo mutants shows significant disparity from wild-type. Among the differences, the defense response genes are elevated in atdxo while photosynthesis-related and plastid genesis-related genes are downregulated. The constitutive expression of defense response genes causes the autoimmune phenotypes of atdxo. This could be modulated by temperature and is partially dependent on the master immunity regulators EDS1 or NPR1. Reactive oxygen species (ROS) accumulation was also detected in the atdxo mutant, and atdxo showed insensitivity to oxidative stress imposed by paraquat. Moreover, the atdxo mutant is hypersensitive to salt stress but not sensitive to general osmotic stress. In Arabidopsis, the 5'-3' RNA decay pathway could act as a repressor of endogenous post-transcriptional gene silencing (PTGS), which is regulated by small RNAs (sRNA). The mutation of AtDXO caused productions of 24- and 25-nucleotide endogenous sRNAs. The growth defect phenotype of atdxo could not be repressed by dysfunction of the RDR6 (RNA-DEPENDENT RNA POLYMERASE 6)-dependent sRNA biogenesis pathway. These findings demonstrate that AtDXO functions as a 5'-3' exoribonuclease both in vitro and in vivo to regulate plant development and to mediate the response to environmental stresses.