Elucidation of the final reactions of DIMBOA-glucoside biosynthesis in maize: characterization of Bx6 and Bx7

Abstract

Benzoxazinoids were identified in the early 1960s as secondary metabolites of the grasses that function as natural pesticides and exhibit allelopathic properties. Benzoxazinoids are synthesized in seedlings and stored as glucosides (glcs); the main aglucone moieties are 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA). The genes of DIBOA-glc biosynthesis have previously been isolated and the enzymatic functions characterized. Here, the enzymes for conversion of DIBOA-glc to DIMBOA-glc are identified. DIBOA-glc is the substrate of the dioxygenase BENZOXAZINLESS6 (BX6) and the produced 2,4,7-trihydroxy-2H-1,4-benzoxazin-3-(4H)-one-glc is metabolized by the methyltransferase BX7 to yield DIMBOA-glc. Both enzymes exhibit moderate K(m) values (below 0.4 mm) and k(cat) values of 2.10 s(-1) and 0.25 s(-1), respectively. Although BX6 uses a glucosylated substrate, our localization studies indicate a cytoplasmic localization of the dioxygenase. Bx6 and Bx7 are highest expressed in seedling tissue, a feature shared with the other Bx genes. At present, Bx6 and Bx7 have no close relatives among the members of their respective gene families. Bx6 and Bx7 map to the cluster of Bx genes on the short arm of chromosome 4.

Figure 1.

Enzymes and intermediates of DIMBOA-glc biosynthesis in maize. Conversion of indole-3-glycerole phosphate to indole by the enzyme BX1 represents the branch point from primary metabolism. The introduction of four oxygen atoms into the indole moiety that yields DIBOA is catalyzed by four cytochrome P450-dependent monooxygenases, BX2 to BX5. The resulting DIBOA is glucosylated by the UDP-glucosyltransferases BX8 and BX9. Hydroxylation in position C-7 of the glc is catalyzed by the 2ODD BX6. The final step of the pathway is the O-methylation of TRIBOA-glc by BX7 to produce DIMBOA-glc.

Figure 2.

Analysis of BX6 enzyme activity using heterologously expressed protein and the substrates DIBOA and DIBOA-glc. Overlay of the HPLC traces. The inserts A, B, and C depict the spectra of the respective main peaks. Thick gray line: enzyme assay using DIBOA-glc as a substrate; the substrate is almost completely converted to TRIBOA-glc (spectrum A). Thick black line: enzyme assay with DIBOA as a substrate (spectrum C); no conversion is detected. Thin black line: incubation of DIBOA-glc under assay conditions without enzyme (spectrum B). Assay conditions were described in “Materials and Methods”; incubation time was 30 min. mAU, Milli absorption units.

Figure 3.

Benzoxazinoids in Bx6 mutant plants. Benzoxazinoids were isolated from mutant and wild-type plants (thick black line, mutant with insertion at position 1; thick gray line, mutant with insertion at position 4; thin black line, wild type; Frey et al., 2003) and analyzed as described in “Materials and Methods.” DIBOA-glc is the predominant benzoxazinoid in the mutant. The spectra of the respective main peaks are displayed in the inserts. A and B, DIBOA-glc. C, DIMBOA-glc. mAU, Milli absorption units.

Figure 4.

Subcellular localization of BX6. The fusion protein BX6part-GFP-BX6 is located in the cytoplasm of the cell. No GFP signal is detected in the vacuole. A and C, Control 2 × 35S promoter-GFP. B and D, BX6part-GFP-BX6 fusion. A and B, Transient expression. C and B, Transgenic plants. The bar equals 10 μm.

Figure 5.

Phylogenetic net of plant OMTs. Enzymes with defined substrates were subjected to the analysis using the program TCoffee for the alignment (Poirot et al., 2003), and the evolutionary network was computed with SplitsTree (Huson and Bryant, 2006). COMTs from different species form a distinct relation group; a second group comprises enzymes (SAGAMT, JAGAMT, IAMT, and BAMT) responsible for methylation of plant hormones (salicylic acid, jasmonic acid). The third cluster includes loosely related OMTs involved in methylation of intermediates of different secondary metabolite pathways. BX7 is linked to this group.

Figure 6.

Expression of DIMBOA-glc biosynthesis genes. Three- and 4-d-old etiolated seedlings of line B73 were dissected in root, scutellar node and mesocotyl (sc), and shoot (leaves and coleoptile); after 6 d, mesocotyl, leaves, and coleoptile were pooled (mshoot). Tissues of older plants were taken after 3 weeks (root) and 10 weeks. Steady-state mRNA levels were measured by real-time reverse transcription-PCR using gene-specific primer pairs. GAP C mRNA quantities were determined in parallel and used for normalization.

Figure 7.

The genes Bx1 to Bx8 are located on the short arm of chromosome 4. Bx7 is about 35 cM distal to Bx6. Bx1 to Bx6 and Bx8 map within 6 cM. The OMT Zrp4 maps at position 49; the maize gene Bm3 is located in position 61.7. The positions are given with respect to the Genetic 2005 map for chromosome 4 by E. Coe (www.maizegdb.org). Bold numbers refer to centiMorgan.