Suberin biosynthesis in <em>O. sativa : </em>characterisation of a cytochrome P450 monooxygenase

In endodermal and exodermal cell walls of roots the polyester suberin forms a radial diffusion barrier for water, solutes and gases. Depolymerization of this otherwise insoluble lipophilic polymer releases its aliphatic monomers, which comprise derivates of very long chain carboxylic acids such as alcohols, fatty acids, ω-hydroxyacids and dicarboxylic acids, as well as the aromatic compounds ferulic and coumaric acid. Based on the chemical composition of suberin some key functions of its biosynthesis have been predicted and first genes could be characterized in the model plant Arabidopsis thaliana in the last decade. In the course of this work, a number of suberin candidate genes could be identified in the globally important crop and model species Oryza sativa based on in silico studies and the analysis of gene expression during physiological conditions promoting suberisation. A reverse genetic approach with focus on cytochrome P450 enzymes led to the discovery of a monooxygenase of very long chain fatty acids in roots. Plants carrying knock out mutations in CYP86B3 were lacking bifunctional aliphatic suberin monomers with carbon chain lengths C24 and higher. Additionally, an accumulation of putative precursors of the affected monomers, the acid methyl esters with corresponding chain lengths, was found in root total lipid extracts of cyp86b3-1. Though no visible phenotype of the mutation was determined after cultivation in hydroponic culture and under osmotic stresses, permeability of diffusion barriers was altered: transport experiments with a photosynthesis inhibitor revealed significantly faster uptake of this apoplastic tracer in roots of cyp86b3-1 impaired in the suberin composition. The transgenic expression of CYP86B3 in A. thaliana could restore the wild type level of ω-hydroxy acids with chain length C22 and C24 in root suberin of the heterologous knock out mutant ralph and ralph/horst