SYSTEMATIC REVERSE GENETICS OF TRANSFER-DNA-TAGGED LINES OF ARABIDOPSIS - ISOLATION OF MUTATIONS IN THE CYTOCHROME-P450 GENE SUPERFAMILY

We have developed an efficient reverse-genetics protocol that uses exp edient pooling and hybridization strategies to identify individual tra nsfer-DNA insertion lines from a collection of 6000 independently tran sformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher p lants P450 genes encode enzymes that perform an exceptionally wide ran ge of functions, including the biosynthesis of primary metabolites nec essary for normal growth and development, the biosynthesis of secondar y products, and the catabolism of xenobiotics. Despite their importanc e, progress in assigning enzymatic function to individual P450 gene pr oducts has been slow. Here we report the isolation of the first 12 suc h lines, including one (CYP83B1-1) that displays a runt phenotype (sma ll plants with hooked leaves), and three insertions in abundantly expr essed genes. The DNAs used in this study are publicly available and ca n be used to systematically isolate mutants in Arabidopsis.