Pectic polysaccharides, a class of plant cell wall polymers, form one of the most complex networks known in nature. Despite their complex structure and their importance in plant biology, little is known about the molecular mechanism of their biosynthesis, modification, and turnover, particularly their structure-function relationship. One way to gain insight into pectin metabolism is the identification of mutants with an altered pectin structure. Those were obtained by a recently developed pectinase-based genetic screen. Arabidopsis thaliana seedlings grown in liquid medium containing pectinase solutions exhibited particular phenotypes: they were dwarfed and slightly chlorotic. However, when genetically different A. thaliana seed populations (random T-DNA insertional populations as well as EMS-mutagenized populations and natural variations) were subjected to this treatment, individuals were identified that exhibit a different visible phenotype compared to wild type or other ecotypes and may thus contain a different pectin structure (pec-mutants). After confirming that the altered phenotype occurs only when the pectinase is present, the EMS mutants were subjected to a detailed cell wall analysis with particular emphasis on pectins. This suite of mutants identified in this study is a valuable resource for further analysis on how the pectin network is regulated, synthesized and modified. Flanking sequences of some of the T-DNA lines have pointed toward several interesting genes, one of which is PEC100. This gene encodes a putative sugar transporter gene, which, based on our data, is implicated in rhamnogalacturonan-I synthesis. The subcellular localization of PEC100 was studied by GFP fusion and this protein was found to be localized to the Golgi apparatus, the organelle where pectin biosynthesis occurs. Arabidopsis ecotype C24 was identified as a susceptible one when grown with pectinases in liquid culture and had a different oligogalacturonide mass profile when compared to ecotype Col-0. Pectic oligosaccharides have been postulated to be signal molecules involved in plant pathogen defense mechanisms. Indeed, C24 showed elevated accumulation of reactive oxygen species upon pectinase elicitation and had altered response to the pathogen Alternaria brassicicola in comparison to Col-0. Using a recombinant inbred line population three major QTLs were identified to be responsible for the susceptibility of C24 to pectinases. In a reverse genetic approach members of the qua2 (putative pectin methyltransferase) family were tested for potential target genes that affect pectin methyl-esterification. The list of these genes was determined by in silico study of the pattern of expression and co-expression of all 34 members of this family resulting in 6 candidate genes. For only for one of the 6 analyzed genes a difference in the oligogalacturonide mass profile was observed in the corresponding knock-out lines, confirming the hypothesis that the methyl-esterification pattern of pectin is fine tuned by members of this gene family. This study of pectic polysaccharides through forward and reverse genetic screens gave new insight into how pectin structure is regulated and modified, and how these modifications could influence pectin mediated signalling and pathogenicity. / Pektin Polysaccharide, eine Klasse pflanzlicher Zellwand Polymere, formen eine der komplexesten natürlichen Strukturen. Trotz seiner immensen Bedeutung in der Biologie der Pflanzen sind die Kenntisse über die molekularen Mechanismen der Pektin Biosynthese, dessen Modifikation und Abbau überraschend gering. Eine Möglichkeit neue Einblicke in den pflanzlichen Pektin Metabolismus zu erhalten, ist die Identifizierung von Mutanten mit veränderter Pektinstruktur. Solche Mutanten konnten durch ein neuatiges Selektionsverfahren gefunden werden. Zieht man Keimlinge der Ackerschmalwand (Arabidopsis thaliana) in Flüssigmedium mit Pektinase an, so lässt sich ein typischer Phänotyp beobachten: Die Pflanzen sind kleinwüchsig und leicht chlorotisch. Diesem Verfahren wurden Populationen verschiedener Genotypen (Insertions Linien, EMS Mutanten, natürlich vorkommende Varianten) ausgesetzt. Auf diese Weise wurden Individuen identifiziert, die gegenüber der Pektinase Behandlung eine verminderte oder erhöhte Resistenz aufweisen, was auf eine veränderte Pektinstruktur hindeutet. Die EMS Mutanten wurden einer detaillierten Zellwand Analyse unterzogen. die so in dieser Arbeit identifizierte Kollektion von Mutanten stellt eine wertvolle Ressource für weitere Forschungsansätze zur Regulation, Biosynthese und Modifikation des Pektins dar. Die Lokalisation der Insertionen in den T-DNA Linien führte zur Identifikation interessanter Gene, zu denen der putative Zuckertransporter PEC100 gehört. Dieses Gen steht vermutlich in Verbindung mit der Synthese von Rhamnogalakturonan-I, einem Bestandteil des Pektins. In dieser Arbeit konnte PEC100 im Golgi Apparat, dem Ort der Pektin Biosynthese, lokalisiert werden. Die natürlich vorkommende Variante C24 ist besonders empfindlich gegenüber der Pektinase. Diese Empfindlichkeit konnte anhand rekombinanter Inzucht Linien auf drei bedeutende quantitative Merkmalsloci (QTL) eingegrenzt werden. C24 zeigte zudem ein gegenüber der Referenz verändertes Massenprofil der Oligogalakturonide. Diese werden derzeit als Signalmoleküle in der pflanzlichen Pathogenabwehr diskutiert, was mit der in dieser Arbeit geseigten Resistenz von C24 gegenüber Schwarzfleckigkeit verursachende Pilz (Alternaria brassicicola) korreliert. In einem revers-genetischen Ansatz wurden zudem Mitglieder der Pektin Methyltransferase Familie als potentielle Enzyme getestet, die die Pektin Methylesterifikation beeinflussen könnten. Diese Mutation in einer dieser Methyltransferasen führte zu Veränderungen des Oligogalakturonid Massenprofils. Dies bestätigt die Hypothese, dass Mitglieder dieser Genfamilie an der Regulation der Methylesterifikation von Pektin beteiligt sind. Die vorliegende Studie, in der ein genetishen Selektionverfahren und Methoden der reversen Genetik kombiniert wurden, hat neue Einblicke in die Regulation und Modifikation von Pektin geliefert.

Over the past few years a large amount of work has provided growing insight into the molecular mechanisms that direct post-Golgi trafficking events in the budding yeast Saccharomyces cerevisae. However, a key event in this process, the formation of secretory vesicles at the Golgi and sorting of cargo into these transport carriers, remains poorly understood. It has been demonstrated that phosphatidylinositol 4-phosphate (PI(4)P) generated by the PI(4)-kinase Pik1p plays an essential role in maintenance of Golgi secretory function and morphology. Up to now relatively few targets of Pik1/PI(4)P signaling at the Golgi have been identified and it thus remains elusive how Pik1p mediates its essential function in Golgi secretion. During my thesis work, I used synthetic genetic array analysis (SGA) of a temperature-sensitive mutant allele of PIK1 (pik1-101) in order to gain better understanding of Pik1p function at the TGN and to isolate new regulators of post-Golgi transport in yeast. I identified a total of 85 genes, whose deletion resulted in a synthetic growth defect when combined with the pik1-101 mutation. 21 isolated deletion mutants were used for further analysis, several of which were found to share common trafficking phenotypes with the pik mutant. A striking result of the screen was the finding that Pik1p interacts genetically with several components of a potential post-translational modification pathway referred to as “urmylation pathway”. In addition, a novel, previously uncharacterized subunit of the Transport protein particle (TRAPP) complex was isolated as genetic interactor of Pik1p, suggesting a function for the TRAPP complex in a Pik1p dependent trafficking pathway. Using tandem affinity purification, I could also demonstrate that TRAPP shows previously unknown interactions with other regulators of post-Golgi transport. The second part of this thesis describes the development of a new visual screening approach. Recent work indicates that secretory cargo in yeast can be transported to the cell surface via at least two different exocytic branches. Upon block of one pathway cargo can be partially redistributed into the other pathway. This partial redundancy of exocytic pathways provides one explanation why genetic screens in the past were largely unsuccessful in identifying the molecular machinery that directs vesicle budding and cargo sorting at the TGN. I collaborated in the development of a novel screening method that was devised to circumvent this problem. The method took advantage of the systematic yeast knockout array and was based on the assumption that a defect in cargo sorting and cell surface transport could be detected as intracellular accumulation of a GFP-tagged model cargo. The suitability of our approach for identifying regulators of secretory transport has been demonstrated in a small-scale pilot study that will be presented in this thesis. The screening method proofed to be applicable on a genome-wide scale and can now be used for the screening of additional markers. This novel approach provides an entry point to the comprehensive study of TGN sorting.

Over the past few years a large amount of work has provided growing insight into the molecular mechanisms that direct post-Golgi trafficking events in the budding yeast Saccharomyces cerevisae. However, a key event in this process, the formation of secretory vesicles at the Golgi and sorting of cargo into these transport carriers, remains poorly understood. It has been demonstrated that phosphatidylinositol 4-phosphate (PI(4)P) generated by the PI(4)-kinase Pik1p plays an essential role in maintenance of Golgi secretory function and morphology. Up to now relatively few targets of Pik1/PI(4)P signaling at the Golgi have been identified and it thus remains elusive how Pik1p mediates its essential function in Golgi secretion. During my thesis work, I used synthetic genetic array analysis (SGA) of a temperature-sensitive mutant allele of PIK1 (pik1-101) in order to gain better understanding of Pik1p function at the TGN and to isolate new regulators of post-Golgi transport in yeast. I identified a total of 85 genes, whose deletion resulted in a synthetic growth defect when combined with the pik1-101 mutation. 21 isolated deletion mutants were used for further analysis, several of which were found to share common trafficking phenotypes with the pik mutant. A striking result of the screen was the finding that Pik1p interacts genetically with several components of a potential post-translational modification pathway referred to as “urmylation pathway”. In addition, a novel, previously uncharacterized subunit of the Transport protein particle (TRAPP) complex was isolated as genetic interactor of Pik1p, suggesting a function for the TRAPP complex in a Pik1p dependent trafficking pathway. Using tandem affinity purification, I could also demonstrate that TRAPP shows previously unknown interactions with other regulators of post-Golgi transport. The second part of this thesis describes the development of a new visual screening approach. Recent work indicates that secretory cargo in yeast can be transported to the cell surface via at least two different exocytic branches. Upon block of one pathway cargo can be partially redistributed into the other pathway. This partial redundancy of exocytic pathways provides one explanation why genetic screens in the past were largely unsuccessful in identifying the molecular machinery that directs vesicle budding and cargo sorting at the TGN. I collaborated in the development of a novel screening method that was devised to circumvent this problem. The method took advantage of the systematic yeast knockout array and was based on the assumption that a defect in cargo sorting and cell surface transport could be detected as intracellular accumulation of a GFP-tagged model cargo. The suitability of our approach for identifying regulators of secretory transport has been demonstrated in a small-scale pilot study that will be presented in this thesis. The screening method proofed to be applicable on a genome-wide scale and can now be used for the screening of additional markers. This novel approach provides an entry point to the comprehensive study of TGN sorting.