Phylogenomics of the benzoxazinoid biosynthetic pathway of Poaceae: gene duplications and origin of the Bx cluster

Abstract

BackgroundThe benzoxazinoids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one (DIMBOA), are key defense compounds present in major agricultural crops such as maize and wheat. Their biosynthesis involves nine enzymes thought to form a linear pathway leading to the storage of DI(M)BOA as glucoside conjugates. Seven of the genes (Bx1-Bx6 and Bx8) form a cluster at the tip of the short arm of maize chromosome 4 that includes four P450 genes (Bx2-5) belonging to the same CYP71C subfamily. The origin of this cluster is unknown.ResultsWe show that the pathway appeared following several duplications of the TSA gene (α-subunit of tryptophan synthase) and of a Bx2-like ancestral CYP71C gene and the recruitment of Bx8 before the radiation of Poaceae. The origins of Bx6 and Bx7 remain unclear. We demonstrate that the Bx2-like CYP71C ancestor was not committed to the benzoxazinoid pathway and that after duplications the Bx2-Bx5 genes were under positive selection on a few sites and underwent functional divergence, leading to the current specific biochemical properties of the enzymes. The absence of synteny between available Poaceae genomes involving the Bx gene regions is in contrast with the conserved synteny in the TSA gene region.ConclusionsThese results demonstrate that rearrangements following duplications of an IGL/TSA gene and of a CYP71C gene probably resulted in the clustering of the new copies (Bx1 and Bx2) at the tip of a chromosome in an ancestor of grasses. Clustering favored cosegregation and tip chromosomal location favored gene rearrangements that allowed the further recruitment of genes to the pathway. These events, a founding event and elongation events, may have been the key to the subsequent evolution of the benzoxazinoid biosynthetic cluster.

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants

“…It is partially conserved in the grass family, which evolved about 50 million years ago (Paterson et al, 2004). Orthologs of the core enzymes Bx1 to Bx5 were found in wheat and rye (Secale cereale), albeit in two separate chromosome regions (Dutartre et al, 2012). Overall, our analysis combined with previously reported information for these characterized plant gene clusters indicate that they evolved in a lineage-specific manner.…”

“…To assess the degree of cluster conservation, we examined nine experimentally characterized metabolic gene clusters that were reported to be conserved in more than one species (Qi et al, 2004;Field et al, 2011;Takos et al, 2011;Dutartre et al, 2012;Itkin et al, 2013;Miyamoto et al, 2016). To characterize the degree of conservation of these clusters, we expanded the analysis to include additional species.…”

Something old, something new: Conserved enzymes and the evolution of novelty in plant specialized metabolism

“…The IGL/BX1 lineage originated from the a-subunit of the Trp synthase complex (TSa; Frey et al, 1997;Melanson et al, 1997) via a single gene duplication event prior to the divergence between maize, wheat (Triticum aestivum), and barley (Hordeum vulgare) and then diversified into IGL and BX1 activities via additional rounds of duplication (Grün et al, 2005;Dutartre et al, 2012). Normally, the TSa subunit is found in a complex with the TSb subunit, making a physical channel that passes indole, the product of TSa, over to TSb.…”

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

“…The 'founding event' of the pathway was suggested to be the clustering of duplicated tryptophan synthase a-subunit (TSA) and an ancestral CYP71C gene, followed by neofunctionalization of both into Bx1 and Bx2, respectively, and yielding biosynthetic access to indole and the oxidized indolin-2-one, derived from the primary metabolite indole-3-glyceraldehyde. The elongation of this cluster through duplication of Bx2 (CYP71C1) giving rise to Bx3 (CYP71C2) was followed by two subsequent duplications of Bx3 resulting in Bx4 (CYP71C3) and Bx5 (CYP71C4), and generated the material that, after neofunctionalization, resulted in a series of successive oxidations from indolin-2-one via 3-hydroxy indolin-2-one and 2-hydroxy-1,4(2H)-benzoxazin-3(4H)-one (HBOA) to 2,4-dihydroxy-2H-1,4-benzoxazin-3 (4H)-one (DIBOA) [104,105]. Both HBOA and DIBOA are subsequently glycosylated by Bx8/Bx9, further hydroxylated by Bx6 (a 2-oxogluterate-dependent dioxygenase) to 2,4,7-trihydroxy-2H-1,4-benzoxazin-3(4H)-one (TRIBOA)-glc and methoxylated by Bx7 to 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA)-glc ( [104] and references therein).…”