Reinventing metabolic pathways: Independent evolution of benzoxazinoids in flowering plants.

Benzoxazinoids (BXDs) form a class of indole-derived specialized plant metabolites with broad antimicrobial and antifeedant properties. Unlike most specialized metabolites, which are typically lineage-specific, BXDs occur sporadically in a number of distantly related plant orders. This observation suggests that BXD biosynthesis arose independently numerous times in the plant kingdom. However, although decades of research in the grasses have led to the elucidation of the BXD pathway in the monocots, the biosynthesis of BXDs in eudicots is unknown. Here, we used a metabolomic and transcriptomic-guided approach, in combination with pathway reconstitution in Nicotiana benthamiana, to identify and characterize the BXD biosynthetic pathways from both Aphelandra squarrosa and Lamium galeobdolon, two phylogenetically distant eudicot species. We show that BXD biosynthesis in A. squarrosa and L. galeobdolon utilize a dual-function flavin-containing monooxygenase in place of two distinct cytochrome P450s, as is the case in the grasses. In addition, we identified evolutionarily unrelated cytochrome P450s, a 2-oxoglutarate-dependent dioxygenase, a UDP-glucosyltransferase, and a methyltransferase that were also recruited into these BXD biosynthetic pathways. Our findings constitute the discovery of BXD pathways in eudicots. Moreover, the biosynthetic enzymes of these pathways clearly demonstrate that BXDs independently arose in the plant kingdom at least three times. The heterogeneous pool of identified BXD enzymes represents a remarkable example of metabolic plasticity, in which BXDs are synthesized according to a similar chemical logic, but with an entirely different set of metabolic enzymes.

The lactonase BxdA mediates metabolic specialisation of maize root bacteria to benzoxazinoids.

Root exudates contain specialised metabolites that shape the plant's root microbiome. How host-specific microbes cope with these bioactive compounds, and how this ability affects root microbiomes, remains largely unknown. We investigated how maize root bacteria metabolise benzoxazinoids, the main specialised metabolites of maize. Diverse and abundant bacteria metabolised the major compound in the maize rhizosphere MBOA (6-methoxybenzoxazolin-2(3H)-one) and formed AMPO (2-amino-7-methoxy-phenoxazin-3-one). AMPO forming bacteria were enriched in the rhizosphere of benzoxazinoid-producing maize and could use MBOA as carbon source. We identified a gene cluster associated with AMPO formation in microbacteria. The first gene in this cluster, bxdA encodes a lactonase that converts MBOA to AMPO in vitro. A deletion mutant of the homologous bxdA genes in the genus Sphingobium, did not form AMPO nor was it able to use MBOA as a carbon source. BxdA was identified in different genera of maize root bacteria. Here we show that plant-specialised metabolites select for metabolisation-competent root bacteria. BxdA represents a benzoxazinoid metabolisation gene whose carriers successfully colonize the maize rhizosphere and thereby shape the plant's chemical environmental footprint.

The wheat dioxygenase BX6 is involved in the formation of benzoxazinoids in planta and contributes to plant defense against insect herbivores.

Benzoxazinoids are plant specialized metabolites with defense properties, highly abundant in wheat (Triticum), one of the world's most important crops. The goal of our study was to characterize dioxygenase BX6 genes in tetraploid and hexaploid wheat genotypes and to elucidate their effects on defense against herbivores. Phylogenetic analysis revealed four BX6 genes in the hexaploid wheat T. aestivum, but only one ortholog was found in the tetraploid (T. turgidum) wild emmer wheat and the cultivated durum wheat. Transcriptome sequencing of durum wheat plants, damaged by either aphids or caterpillars, revealed that several BX genes, including TtBX6, were upregulated upon caterpillar feeding, relative to the undamaged control plants. A virus-induced gene silencing approach was used to reduce the expression of BX6 in T. aestivum plants, which exhibited both reduced transcript levels and reduced accumulation of different benzoxazinoids. To elucidate the effect of BX6 on plant defense, bioassays with different herbivores feeding on BX6-silenced leaves were conducted. The results showed that plants with silenced BX6 were more susceptible to aphids and the two-spotted spider mite than the control. Overall, our study indicates that wheat BX6 is involved in benzoxazinoid formation in planta and contributes to plant resistance against insect herbivores.