Prohexadion-Ca (calcium 3-oxido-4-propionyl-5-oxo-3-cyclohexen-carbonic acid) is a specific inhibitor of 2-oxoglutarate-dependent dioxygenases (Rademacher et al., In: Karssen, C. M., van Loon, L. C., Vreugdenhil, D. (Eds.), Progress in Plant Growth Regulation. Kluwer Academic Publishers, Amsterdam, pp. 571-577, 1992). To investigate the participation of this type of oxygenase in the biosynthesis of DIMBOA, seedlings were grown in the presence of prohexadion-Ca. The inhibition of 2-oxoglutarate-dependent dioxygenases by prohexadion-Ca leads to an arrest of the pathway at DIBOA and an almost complete loss of DIMBOA in the seedlings (Figure 1). Hence, the hydroxylation of DIBOA at position 7 is most probably catalyzed by a 2-oxoglutarate-dependent dioxygenase.
The maize EST-data collection of Pioneer Hi-Bred was screened for the presence of 2-oxoglutarate-dependent-dioxygenase sequences. Ten genes were found predominantly in EST-libraries derived from seedling material, the main tissue of DIMBOA biosynthesis. Full-size cDNA clones were retrieved for candidate genes and assayed by northern analysis. One 2-oxoglutarate-dependent-dioxygenase gene displayed co-expression with previously identified bx genes.
A reverse genetic approach was taken in order to verify the function of this 2-oxoglutarate-dependent dioxygenase named bx6 (GenBank Accession number AF540907). Comparison with the genomic sequence revealed that bx6 is a gene without introns. Four independent integrations of the transposable element Mutator (Mu) that are scattered across the cDNA sequence were uncovered in the TUSC population. Four maize lines representing the different Mu-integration events were analyzed for the presence of benzoxazinoids. Mutant individuals that no longer synthesize DIMBOA but include DIBOA as the main benzoxazinoid were identified in the F2 progeny of three lines. A total of 42 plants were analyzed, and in all cases, a wildtype phenotype was connected with the presence of at least one wildtype allele of the dioxygenase gene, and the mutant phenotype was displayed only by plants that were homozygous for the integration of a Mu-element into bx6. This co-segregation provides evidence that the gene responsible for the hydroxylation of DIBOA, bx6, in maize has been identified.
A distinct feature of the bx-genes of maize is the clustering on the short arm of chromosome 4 (Gierl and Frey, 2001). Therefore, the location of the bx6 gene in the maize genome is an interesting feature. Mapping of bx6 using the recombinant inbred population CM37xT232 (Burr and Burr, Trends Genet. 7:55-61, 1991) places bx6 in close proximity of the other of bx genes on chromosome 4. bx6 and bx3/bx4 are separated by 7 centiMorgan. These results are in good accordance with previous mapping data for the hydroxylation of the C-7 position in DIMBOA-biosynthesis (K. Lobos and P. Sisco, personal communication).
Benzoxazinoids are widely distributed in grasses. The P450 enzymes that convert indole to DIBOA are functionally conserved within cereal species (Glawischnig et al., Phytochemistry 50:925-930, 1999; Nomura et al., Mol. Gen. Genet. 267:210-217, 2002). Prohexadion-Ca incubation inhibits DIMBOA formation in wheat (Figure 1). This indicates that this enzymatic function is also conserved in cereals.
1. A 2-oxoglutarate-dependant dioxygenase is necessary for DIMBOA biosynthesis
in maize and wheat. A, B Analysis of benzoxazinones isolated from
4 day old etiolated maize seedlings (A) or wheat seedlings (B) grown in
water. The main substance is DIMBOA. C, D Maize seedlings (C) or
wheat seedlings (D) were grown 4 days etiolated in the presence of the
inhibitor prohexadion-Ca. The main benzoxazinone is DIBOA.
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