Outside the Poales, benzoxazinoids are detected in two distant orders of the eudicots, the Ranunculales and the Lamiales (Figure 2). In contrast to the situation in the Poales, benzoxazinoid biosynthesis in these orders is restricted to single isolated species.


Research Interest: Secondary metabolism: Benzoxazinoid biosynthesis

At least 200.000 secondary metabolites are found in the plant kingdom. Many of these natural products are specialised metabolites that are produced only by certain taxa. Benzoxazinoids represent protective (Figure 1) and allelophatic metabolites that are found in a multitude of species of the family Poaceae (Gramineae) of the monocot plants, including the major agricultural crops maize, wheat and rye. Benzoxazinoids confer protection against a wide range of herbivores, pathogenic fungi and bacteria.











Benzoxazinoid biosynthesis in maize and wild barley has been elucidated by our group (Frey et al., 1997; 2003; v. Rad et al., 2001; Jonczyk et al., 2008; Grün et al., 2005; Figure 3).  


Benzoxazinoid biosynthesis

The branchpoint from the primary metabolism (Bx1 gene) can be traced back to duplication and neo-functionalisation of the alpha-subunit of tryptophan synthase (TSA) (Figure 4).

Modification of the intermediates by consecutive hydroxylation is catalysed by members of a cytochrome P450 enzyme subfamily (Bx2-Bx5). Glucosylation by an UDP-glucosyltransferase (UGT, Bx8, Bx9) is essential for the reduction of autotoxicity of the benzoxazinoids. The glucoside is stored in the vacuole; the specific glucosidase is located in the plastid (monocots) or in the apoplast (dicots).

Upon disintegration of the cell due to pathogen or pest attack this compartimentisation is broken and the ß-glucosidase bioactivates the defence metabolite. 

In the Poaceae, the expression of the pathway is developmentally regulated and highest benzoxazinoid levels are displayed at about four days after germination, both in root and shoot. In this stage, the DIMBOA concentration exceeds the total tryptophan concentration in maize by a factor of 20 (Frey et al., 1997).

In maize, the genes of the pathway are linked on the short arm of chromosome 4 (Figure 5). The close neighbouring of the Bx genes is an extraordinary feature; enzymes belonging to five different functional classes are assembled in the Bx cluster in maize. 


Benzoxazinoid transport in relation to exudation (Dr. Claudiu Niculaes)

Root exudation is the process by which plant roots secrete chemicals (including benzoxazinoids) into the soil. Exudation is critical in mediating plant-plant and plant-microbe interactions, but the mechanisms by which benzoxazinoids are exported from the roots into the surrounding soil are unknown. We are employing QTL mapping and a pharmacological approach to study benzoxazinoid transport in relation to exudation.




Evolution of secondary metabolic pathways in plants

Three benzoxazinoid producing species, C. orientalis (larkspur), L. galeobdolon (yellow archangel) and A. squarrosa (zebra plant), from two families (Ranunculaceae and Lamiaceae) have been characterised with respect to the benzoxazinoid biosynthetic pathway. It has been shown that, like in the grasses, indole is the first committed intermediate of the pathway. For each of these plants at least one homolog of TSA-genes (Igls) was detected (Schullehner et al., 2008). However, only the C. orientalis enzyme CoBX1 (Figure 4) has similar catalytic properties as the maize and wheat BX1 enzymes. A phylogenetic analysis of TSA homologous enzymes from maize and A. thaliana, maize, C. orientalis, L. galeobdolon and A. squarrosa, shows that the branchpoint enzymes of benzoxazinoid biosynthesis have evolved independently (Schullehner et al., 2008). C. orientalis and L. galeobdolon microsomes use indole as a substrate to produce indolin-2-one in a strictly NADPH dependent manner (Schullehner et al., 2008). This is an indication that the second reaction of DIBOA biosynthesis in dicots is accomplished in a similar manner as in the grasses. Like in the grasses, an UDP-glucosyltransferase catalyses benzoxazionoid-glucoside formation. Hence, benzoxazinoid biosynthesis would, in principle, appear to follow the same chemical route in dicots and monocots. The question whether monophyletic or independent evolution underlies this analogy will be answered by the characterization of dicot genes of the pathway.


At present, the focus of our research is the elucidation of the evolution of secondary metabolic pathways in plants and the characterization of regulatory factors and elements of benzoxazinoid biosynthesis in maize.