Markus Pauly Research Interests continued
Analytical Platform for the Microanalysis of Wall Polysaccharides
Currently mainly classical carbohydrate chemistry based methods are utilized in our lab to describe the structure of particular wall polysaccharides. These methods encompass solubilization of various wall polymers using sequential extraction procedures that make use of wall degrading enzymes and chemicals. The resulting fractions are then analysed using techniques such as monosaccharide composition and glycosidic linkage analysis. In addition, the presence of ester substituents (such as O-acetyl-substituents) is determined. However, such an analysis is rather labor-intensive and time consuming. Therefore, an oligosaccharide mass profiling method (OLIMP) using specific polysaccharide hydrolases in combination with mass spectrometry has been developed. The sensitivity of OLIMP allows for the rapid assessment of even minute amount of tissue-materials. A profile can be obtained from preparations of as little as 500 Arabidopsis cells prepared by a laser-dissection catapulting instrument.
Forward Genetic Approach: Identification of Structural Wall Mutants
One mean to identify wall mutants is the screening of populations of chemically mutagenised Arabidopsis seeds for novel structural wall mutants using OLIMP. This approach takes advantage of the speed of OLIMP and has lead to the identification of 60 distinct mutants with altered xyloglucan structures including the abundance of ester-substituents. Map-based cloning of the mutated genes should give valuable insights into biosynthesis, metabolism and function of structural variations of xyloglucan.
Chemical Genetics with Specific Glycosylhydrolases
An additional approach is based on chemical genetics. Arabidopsis seeds can be germinated and grown in liquid culture with the addition of agents such as specific polysaccharide hydrolases. Due to the presence of the enzyme the seedlings exhibit distinct phenotypes (e.g., alterations in stomata development, growth and hypersensitive response phenotypes) that can be reversed when the agent, i.e. the enzyme, is removed. The enzyme containing medium was employed to screen mutagenized or T-DNA tagged Arabidopsis seed populations for the identification of mutants that exhibit a more severe phenotype ("hypersensitive mutants") or resembling the wild type phenotype grown without the enzyme ("resistant mutants"). Indeed, several mutants identified by their altered visible phenotype when subjected to the hydrolase exhibit an altered wall structure. Further characterization of the mutant phenotype as well as understanding the genetic basis for the phenotype should result not only in additional mutants with novel wall structures but also mutants impaired or altered in oligosaccharin signalling pathways.Reverse Genetic Approach: Cell Wall Biosynthesis
Although information about the structural components of cell walls has considerably increased in recent years, very little is known about the biosynthesis of individual wall components on a molecular level. Thus, a reverse genetic approach is employed after the identification of candidate genes involved in this process. Currently, numerous novel genes involved in the synthesis of nucleotide sugars, the substrates for polysaccharide synthesis, have been identified through bioinformatic means by comparison to gene-sequences of well-characterized bacterial enzymes. The analysis of Arabidopsis insertional knock-out alleles of these candidate genes has already revealed, in some cases, a function for the gene (e.g., a UDP-rhamnose synthase), a plant with an altered polysaccharide composition (e.g., drastic reduction of rhamnogalacturonan I), and its effect on plant growth and morphology (e.g., detrimental seed development and morphology).