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[email protected] Profiling and localization of phenolics in Arabidopsis lignin mutants Oana Dima, Kris Morreel, Ruben Vanholme, Bartel Vanholme and Wout Boerjan VIB Department of Plant Systems Biology; UGent Department of Plant biotechnology and Genetics, Technologiepaerk 927 - 9052 Gent, Belgium Lignin, an aromatic polymer in the cell wall, allows plants to grow upward and to transport water and solutes through the vascular system. However, lignin obstructs agro-industrial processes such as the production of pulp and paper or biofuels from wood. A more easily degradable lignin structure, e.g. by introducing easily cleavable units, would alleviate these problems. This could be done by rerouting metabolites that can potentially co-polymerize into lignin, to the cell wall. We profiled the phenolics in a set of Arabidopsis lignin mutants. Additionally, because most phenolics are transported to the apoplast or the vacuole, we are in the process of profiling the protoplasts and the vacuoles isolated from these mutants to reveal the subcellular site of accumulation of these phenolics. CCR phenolic profiling Comparative leaf profiling Arabidopsis WT and 7 lignin mutants Phenolic profiling FT-ICR-MS based identification Subcellular localization of interesting compounds Vacuole isolation vacuole buffer 4% ficoll interphase with purified vacuoles protoplasts in lysis buffer with 10% ficoll 35 days old Arabidopsis rosette leaves (2 grams) 4h with cellulase and macerozyme Washing cell walls and tissue away from protoplasts Lysis of protoplasts and ficoll gradient GFP Col-0/FM4-64 GFP/FM4-64 Phenolic extraction SPE steps Phenolic profiling LLE vs SPE Glucosinolates 12.3 2.9 12.4 Load 3.0 4.0 4.3 LLE 2.8 9.7 11.0 13.9 16.3 3.2 Wash 4.3 7.1 6.5 5.9 13.2 7.3 9.8 11.5 14.1 15.5 Protoplasts 26.3 19.4 20.0 22.4 10.5 2.6 30.4 4.1 4.4 5.1 2.0 SPE 11.8 9.2 9.9 5.2 24.2 15.4 28.2 32.4 26.9 30.4 13.7 9.9 3.1 2.5 Leaves 19.8 3.1 2.9 3.3 Sinapoyl malates Kaempherol glycosides 13.1 5.9 6.5 7.1 2.2 Ultracentrifugation 4.0 8.5 8.1 Elution 13.3 14.5 19.6 19.7 19.0 16.6 17.4 26.5 22.1 24.4 29.6 29.3 30.8 31.4 32.8 8.5 9.9 3.1 4.0 19.7 20.5 11.4 19.6 12.9 Vacuoles 29.6 13.7 14.5 22.8 11.4 26.9 22.8 16.6 21.1 26.5 24.4 29.2 28.5 30.8 31.4 32.8 Conclusion Perturbation of the lignin biosynthesis pathway in Arabidopsis affects flavonoid and glucosinolate metabolism. Furthermore, several of the accumulating aromatic compounds are candidate lignin monomers. To trace their localization, the phenolic profiling of vacuoles was optimized. Currently, this data is being explored. Profiling of CCR vacuoles