Root branching allows plants to explore rhizosphere, to gain efficiently water, mineral nutrients or enter in various biotic interactions. Initiation of lateral root formation is localized to pericycle cells, flanking the xylem poles of diarch vascular bundle in Arabidopsis thaliana. Right in these pericycle cells, there is the expression pattern of the gene trap line MGT180. In this theseis , I have provided the evidence that the expression pattern of MGT180 is related with AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED18 (AHL18; At3g60870). AHL18 belongs to a gene family of 29 transcriptional factors of Arabidopsis. AHL18 has not been functionally characterized yet, the analysis of singlemutant ahl18 and some others revealed no significant phenotype. However, one of doublemutants, E15, showed a significant phenotype. This phenotype was evident mainly in the aboveground part of plants, and was not corresponding to any phenotype of AHL mutation described so far. There is a known redundancy among some AHL genes, confirmed by crossing of ahl18 and ahl28 leading to E15 plant. Translation phusion AHL18-mRUBY and AHL22-mRUBY under native promotors should reveal, where these two related proteins act, and if they fiction in autonomous manner or not. Key words Arabidopsis thaliana, lateral root, AHL, pericycle

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.

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...

Philosophiae Doctor - PhD / In this thesis, we have firstly cloned and expressed the domains that harbours the putative catalytic GC domain in these receptor molecules and demonstrate that these molecules can convert GTP to cGMP in vitro. Secondly, we show that exogenous application of both Phytosulfokine and Brassinosteroid increase changes of intracellular cGMP levels in Arabidopsis mesophyll protoplast demonstrating that these molecules have GC activity in vivo and therefore provide a link as second messenger between the hormones and down-stream responses. In order to elucidate a relationship between the kinase and GC domains of the PSK receptor, we have used the AtPSKR1 receptor as a model and show that it has Serine/Threonine kinase activity using the Ser/Thr peptide 1 as a substrate. In addition, we show that the receptor`s ability to phosphorylate a substrate is affected by the product (cGMP) of its co-domain (GC) and that the receptor autophosphorylates on serine residues and this step was also observed to be affected by cGMP. When Arabidopsis plants are treated with a cell permeable analogue of cGMP, we note that this can affect changes in the phosphoproteome in Arabidopsis and conclude therefore that the cGMP plays a role in kinase-dependent downstream signalling. The obtained results suggest that the receptor molecules investigated here belong to a novel class of GCs that contains both a cytosolic kinase and GC domains, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors NPR1 and NPR2. The findings also strongly suggest that cGMP has a role as a second messenger in both Brassinosteroid and Phytosulfokine signalling. We speculate that other proteins with similar domain organisations may also have dual catalytic activities and that a significant number of GCs, both in plants and animals, remain to be discovered and characterised. / South Africa

Chitinases are a diverse family of proteins occurring in plants. Their function varies considerably, with certain chitinases having been associated with development. The majority however, are pathogenesis related (PR) proteins that have been shown to play a role during plant pathogen interactions. This has lead to many investigations on the use of chitinases in providing transgenic disease resistance. These studies are usually done using a constitutive expression system. This however stands in contrast with the natural defense system were PR gene expression is usually only upregulated when the plant is exposed to abiotic and/or biotic stress factors. The constitutive expression is therefore not ideal as it increases ‘cost’ penalties due to the energy being spent expressing the gene. In this study however, an inducible expression system was applied using a stress inducible promoter AtGSTF6 derived from Arabidopsis thaliana, to drive Lupinus albus IF3 chitinase expression when the plants are under pathogen attack. The construct AtGSTF6-IF3 was inserted into the binary vector pCAMBIA 2300 and transformed into Nicotiana Tabacum cv JR6 by Agrobacterium-mediated transformation. To demonstrate the functionality of such a construct, an expression study was done on transgenic N. Tabacum to determine transcription and in vitro chitinase enzyme activity. The data revealed that IF3 chitinase gene transcription from lupin plants was achieved in N. Tabacum. Nine of the twelve lines that tested positive for chitinase gene transcription after hydrogen peroxide treatment, showed increased chitinase activity. With the success of showing increased chitinase activity, these lines were subjected to a detached leaf assay with Rhizoctonia solani AG2, which causes leaf target spot disease. The assay showed that six of the nine lines identified as having increased chitinase activity showed reductions in lesion areas. More specifically, three of the four lines showing more than a five-fold increase in chitinase activity compared to the untransformed N. Tabacum, showed significant lesion reduction. The AtGSTF6-IF3 construct can therefore be recommended to increase disease resistance in N. Tabacum towards Rhizoctonia solani AG2 after showing both expression and increased disease resistance in certain transgenic lines. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

Les bactéries phytopathogènes produisent des effecteurs de type III qui sont des facteurs de virulence injectés dans la cellule hôte afin de moduler les défenses et ainsi favoriser l'infection. L'effecteur PopP2 de Ralstonia solanacearum possède une activité acétyl-transférase qui est perçue par la paire de protéines de résistance RRS1-R et RPS4 dans le noyau des cellules végétales. Cette étape de perception conduit à l'activation des réponses de défenses. GTE9 et GTE11, deux protéines à bromodomaine d'Arabidopsis thaliana, s'apparentent à des lecteurs épigénétiques ciblés par PopP2. Nos données démontrent que ces protéines GTEs (i) interagissent avec PopP2 dans le noyau des cellules végétales et (ii) sont acétylées en présence de l'effecteur. De plus, GTE9 et GTE11 se lient in vivo et in vitro à des histones H4, suggérant que PopP2 interfère avec des processus de remodelage de la chromatine. / Microbial pathogens infect host cells by delivering virulence factors (effectors) that interfere with defenses, thereby favouring infection. The Ralstonia solanacearum PopP2 effector displays an acetyltransferase activity perceived by the Arabidopsis immune receptor pair, RRS1-R with RPS4, in the plant nucleus. This recognition step leads to the activation of immunity. Two Arabidopsis bromodomain-containing proteins , called General Transcription factor with Extra-terminal domain 9 and 11 (GTE9 and GTE11), were identified as PopP2-interacting partners. GTE9 and GTE11 are considered as epigenetic readers. Our data demonstrate that these GTEs proteins (i) interact with PopP2 in the plant nucleus and (ii) are acetylated in presence of the effector. Moreover, bromodomains of GTE9 and GTE11 bind in vivo and in vitro to Histone H4. Together, these data suggest that PopP2, through the targeting of GTE9 and GTE11, likely interfere with chromatin remodeling processes to affect ETS and/or ETI-related processes.

No description available.

How patterns arise from an apparently uniform group of cells is one of the classical problems in developmental biology. The mechanism is complicated by the fact that patterning occurs on a growing medium. Therefore, changes in an organism’s size and shape affect the patterning processes. In turn, patterning itself may affect growth. This interaction between growth and patterning leads to the generation of complex shapes and structures from simpler ones. Studying such interactions requires the possibility to monitor both processes in vivo. To this end, we developed a new technique to monitor and quantify vein patterning in a growing leaf over time using the leaves of Arabidopsis thaliana as a model system. We used a transgenic line with fluorescent markers associated with the venation. Individual leaves are followed in many samples in vivo through time-lapse imaging. Custom-made software allowed us to extract the leaf surface and vein pattern from images of each leaf at each time point. Then average spatial maps from multiple samples that were generated revealed spatio-temporal gradients. Our quantitative description of wild type vein patterns during leaf development revealed that there is no constant size at which a part of tissue enclosed by vasculature will become irrigated by a new vein. Instead, it seemed that vein formation depends on the growth rate of the tissue. This is the first time that vein patterning in growing leaves was quantified. The techniques developed will later be used to explore the interaction between growth and patterning through a variety of approaches, including mutant analysis, pharmacological treatments and variation of environmental conditions.

Chez les eucaryotes, les gènes d’ARNr 45S sont présents en un grand nombre de copies organisées dans des régions chromosomiques appelées NOR pour « Nucleolus Organizer Region ». Cependant, seule une fraction de ces gènes est activement exprimée et leur activation/répression est majoritairement contrôlée par des mécanismes épigénétiques. Parmi les facteurs requis pour l’expression de ces gènes, se trouve la nucléoline, une protéine majeure du nucléole. Chez A. thaliana, la protéine NUC1 est nécessaire pour le maintien de la méthylation et le control de l’expression de variants spécifiques des gènes d’ARNr. De manière intéressante, contrairement aux animaux et aux levures, le génome des plantes possède un deuxième gène codant la nucléoline : NUC2. Au cours de cette étude, nous avons montré que les deux gènes NUC1 et NUC2 sont nécessaires pour la survie de la plante. L’étude de plantes mutées pour le gène NUC2 a révélé que cette protéine est impliquée dans l’organisation et l’expression des ADNr mais par des mécanismes antagonistes à ceux de son homologue NUC1. En effet, l’absence de la NUC2 induit une hyperméthylation des ADNr ainsi qu’une réorganisation spatiale et une variation du nombre de copie des différents variants des gènes d’ARNr. Par ailleurs, la protéine NUC1 se lie aux gènes actifs alors que la protéine NUC2 est associée à la chromatine condensée en périphérie du nucléole. En parallèle, nous avons montré que l’expression des ADNr est affectée en réponse à la chaleur et que le gène NUC2 est fortement induit. L’ensemble de ces données suggèrent un potentiel rôle de la NUC2 dans la répression des gènes d’ARNr au cours du développement et en réponse au stress. / In eukaryotes, 45S rRNA genes are highly repeated and localize in chromosomal regions known as NOR for “Nucleolus Organizer Regions”. However, only a small proportion of these genes is transcriptionally active and their activation and/or repression depends on epigenetic mechanisms. One of the factors involved in rDNA expression is nucleolin, a major nucleolar protein. In A. thaliana, nucleolin protein NUC1 is required to maintain rDNA methylation and control expression of specific rDNA variants. Interestingly, in contrast to animals and yeast, plants encode a second nucleolin gene: NUC2. Here, we show that NUC1 and NUC2 genes are both required for plant survival. Analysis of nuc2 mutant plants reveals that NUC2 protein is required for rDNA organization and expression but with mechanisms antagonistic to those described for its homologue NUC1. In fact, loss of NUC2 induces rDNA hypermethylation and a spatial reorganization of rRNA genes with changes in copy numbers of rDNA variants. Moreover, NUC1 protein binds transcriptionally active rRNA genes while NUC2 protein associates with condensed chromatin in the periphery of the nucleolus. Furthermore, we show that rRNA gene expression is affected in response to heat shock and that the NUC2 gene is strongly induced. Altogether, our results suggest a potential role of NUC2 protein in rDNA repression during development and/or in response to stress.

Acclimation of photosynthesis allows plants to adjust the composition of their photosynthetic apparatus to adapt to changes in the environment, and is important in maintaining fitness. Dynamic acclimation refers to acclimation of fully developed leaves, after developmental processes have ceased. Rates of photosynthesis fluctuate with environmental change, and this requires metabolic adjustments. It has previously been shown that acclimation requires the chloroplastic glucose 6-phosphate/ phosphate translocator GPT2. Using label-free proteomics we characterised the acclimation deficient gpt2 mutant. High light acclimation involves changes in the composition of the photosynthetic proteome and increases in many other metabolic enzymes, but in gpt2 plants, a reduced ability to alter protein composition, and enhanced stress responses were seen. Using a combined transcriptomics and proteomics approach we also analysed acclimation to low temperature. We show that photosynthetic acclimation requires the cytosolic fumarase, FUM2. In fum2 mutants, an enhanced transcriptional response to low temperature was seen, which was impaired at the level of the proteome, relative to the WT. We also identified a protein encoding a β-Amylase, BAM5, that strongly responded to high light acclimation. The role of this protein was further characterised, and we show a nonchloroplastic location. Furthermore, suppression of this gene resulted in plants that were unable to acclimate, and had a reduced sugar content. This research highlights novel and diverse roles for proteins in acclimation, and provides a comprehensive proteomic profiling of high light and low temperature acclimation that has previously been lacking.