Systematic reverse genetics of transfer-DNA-tagged lines of Arabidopsis. Isolation of mutations in the cytochrome p450 gene superfamily

Abstract

We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed 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 plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.

Figure 1

A, To generate the DNAs necessary for this strategy, the seeds were first combined into subpools. Seeds from the first 10 lines (1–10), the next 10 lines (11–20), etc., through 6000 lines were pooled to form 600 subpools. B, For each thousand lines (Sheet Pool), the DNAs from the 100 subpools were mixed in a multiplex array, to give 10 Pooled Column (PC) and 10 Pooled Row (PR) pools. The pooled column and pooled row pools from all of the sheet pools were then amalgamated to give 10 Column Superpools and 10 Row Superpools. When the sheet pool, column superpools, and row superpools containing the positive amplicon are known, it is possible to define the mutant of interest to a subpool of 10 lines.

Figure 2

Summary of the approximate positions of the T-DNA insertions in the P450 genes. A prototypical P450 gene with just one intron represented is shown (UTR, untranslated region; HBR, conserved heme-binding region). The insertions, numbered from 1 to 13, are described in Table I. The directions of the flags indicate the orientations of the border primers relative to the GSPs used to identify the insertion.

Figure 3

The phenotypes of 10-d-old soil-grown plants: left, wild type; right, homozygote containing the CYP83B1-1 mutant allele.