Identification of CYP79B2 and CYP79B3. (A) Yeast expressing CYP79B2 are resistant to 5FI. A saturated culture (0.1 ml) of diploid strain L5140 × L5141 carrying either CYP79B2 or the library vector was mixed with 3.7 ml of molten 0.7% agar and plated onto complete minimal medium lacking uracil and containing 2% galactose and 2 mg/liter Trp. After the top agar had solidified, sterile filter disks were placed onto the surface and 10 μl of the indicated concentration of 5FI was pipetted onto the filter. Yeast plates were incubated for 48 h and photographed. (B) Alignments of deduced amino acid sequences of the CYP79B2 and CYP79B3 proteins and the Sorghum CYP79A1 protein. Sequences were aligned with the program megalign (DNAstar, Madison, WI) by using the Clustal method (24). Amino acid residues identical in at least two of three sequences are boxed, and hyphens indicate gaps introduced to maximize alignment. GenBank accession numbers for the previously identified sequences are: CYP79B2, ATT5J17.120; CYP79B3, AC006592.4; and CYP79A1, U32624. Sequences for the newly isolated cDNAs of CYP79B2 and CYP79B3 are identical to the previously identified sequences.

CYP79B2 and CYP79B3 convert Trp to IAOx. (A) CYP79B2 metabolizes Trp. E. coli membrane extracts carrying CYP79B2mod or a non-P450 cDNA (control) were assayed for Trp-metabolizing activity as described in Materials and Methods. Radioactive compounds derived from [¹⁴C]Trp were detected by using a Bio-Rad Molecular Imager. The positions of Trp and the reaction product (arrow) are indicated. Reaction times are indicated in minutes. (B) IAOx comigrates with the CYP79B2mod reaction product. Similar samples as in A were assayed for Trp-metabolizing activity except that nonradioactive Trp was substituted for [¹⁴C]Trp. Indolic compounds were visualized by UV epifluorescence by using a Bio-Rad Fluor-S imager. Positive images were inverted into negative images by using Adobe photoshop le 3.0. Images were cropped to exclude nonessential portions. IAOx was loaded as a standard. Reaction products are indicated by an arrow, and reaction times are indicated in minutes. (C) CYP79B3 has an activity similar to CYP79B2. Membrane extracts prepared from E. coli expressing CYP79B3, CYP79B2, or the pBluescript KS(+) vector were assayed for Trp-metabolizing activity as in A, and the reactions were visualized as in B. Reaction products are indicated by an arrow, and reaction times are indicated in minutes.

35S-CYP79B2 plants are resistant to toxic analogs of Trp and its precursors. 35S-CYP79B2 and Col-0 plants were germinated and grown for 10 days on PNS medium (30) containing no supplement (A), 10 μM 5MA (B), 15 μM 5MT (C), or 10 μM 5FI (D). Plants were positioned horizontally on fresh, unsupplemented PNS medium for photography.

Induction of the CYP79B2 mRNA by a virulent bacterial pathogen. RNA samples were prepared at times indicated after Arabidopsis was infected with P. syringae pv maculicola (ES4326). A Northern blot prepared from these RNA samples was probed with the CYP79B2 cDNA or β-tubulin. The blot was exposed for 7 days (CYP79B2) or 24 h (β-tubulin).

Trp secondary metabolism derived from IAOx. Cloned genes and corresponding activities for the conversion of Trp to IAOx and for conversion of IAN to IAA are shown. The steps for glucosinolate biosynthesis and other indole glucosinolates are described in ref. 1.