Biosynthesis and chemical transformation of benzoxazinoids in rye during seed germination and the identification of a rye Bx6-like gene.

Benzoxazinoids are secondary metabolites with plant defense properties and possible health-promoting effects in humans. In this study, the transcriptional activity of ScBx genes (ScBx1-ScBx5; ScBx6-like), involved in benzoxazinoid biosynthesis, was analyzed during germination and early seedling development in rye. Our results showed that ScBx genes had highest levels of expression at 24-30 h after germination, followed by a decrease at later stages. For ScBx1-ScBx5 genes expression was higher in shoots compared with root tissues and vice versa for ScBx6-like gene transcripts. Moreover, methylated forms of benzoxazinoids accumulated in roots rather than in shoots during seedling development, in particular reaching high levels of HMBOA-glc in roots. Chemical profiles of benzoxazinoid accumulation in the developing seedling reflected the combined effects of de novo biosynthesis of the compounds as well as the turnover of compounds either pre-stored in the embryo or de novo biosynthesized. Bioinformatic analysis, together with the differential distribution of ScBx6-like transcripts in root and shoot tissues, suggested the presence of a ZmBx6 homolog encoding a 2-oxoglutarate dependent dehydrogenase in rye. The ScBx6-like cDNA was expressed in E. coli for functional characterization in vitro. LC-MS/MS analysis showed that the purified enzyme was responsible for the oxidation of DIBOA-glc into TRIBOA-glc, strongly suggesting the ScBX6-like enzyme in rye to be a functional ortholog of maize ZmBX6.