Biosynthesis and chemical transformation of benzoxazinoids in rye during seed germination and the identification of a rye Bx6-like gene Fariha Tanwir a , Giuseppe Dionisio a , Khem B. Adhikari b , Inge S. Fomsgaard b , Per L. Gregersen a, * a Department of Molecular Biology and Genetics, Aarhus University, Denmark b Department of Agroecology, Aarhus University, Denmark article info Article history: Received 10 March 2017 Received in revised form 20 April 2017 Accepted 24 April 2017 Chemical compounds: DIBOA (PubChem CID: 28495) DIBOA-glc (PubChem CID: 5316752) DIMBOA (PubChem CID: 2358) DIMBOA-glc (PubChem CID: 441563) HBOA (PubChem CID: 322636) MBOA (PubChem CID: 10772) HMBOA (PubChem CID: 152213) TRIBOA (PubChem CID: 10965465) TRIBOA-glc (PubChem CID: 46174045) Keywords: Secale cereale Poaceae Seedling development Benzoxazinoids ScBX6-like cDNA Functional ortholog 2-Oxoglutarate-dependent dehydrogenase abstract 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 develop- ment in rye. Our results showed that ScBx genes had highest levels of expression at 24e30 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. Bio- informatic 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 dehydroge- nase 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. © 2017 Elsevier Ltd. All rights reserved. 1. Introduction Benzoxazinoids, a specialized group of defense and allelopathic compounds in plants, have been studied in a few dicots and several monocots such as maize (Zea mays), wheat (Triticum aestivum), rye (Secale cereale), and wild barley (Hordeum spp.) (Etzerodt et al., 2008; Schullehner et al., 2008; Søltoft et al., 2008; Niemeyer, 2009). Benzoxazinoids are characterized as hetero-aromatic me- tabolites that are categorized into three groups: 1) benzox- azolinones (benzoxazolin-2-one, BOA; and its 6-methoxy deriva- tive, MBOA), 2) lactams (2-hydroxy-1,4-benzoxazin-3-one, HBOA; its 7-methoxy derivative, HMBOA and their glycosides), and 3) hydroxamic acids (2,4-dihydroxy-1,4-benzoxazin-3-one, DIBOA; its 7-methoxy derivative, DIMBOA, and their glycosides). Table 1 Abbreviations: 2-ODD, 2-oxoglutarate dependent dehydrogenase; BOA, 2- benzoxazolinone; Ct, Cycle threshold; DIBOA, 2,4-dihydroxy-1,4-benzoxazin-3- one; DIBOA-glc, 2-b-D glucopyranosyl-4-hydroxy-1,4 benzoxazin-3-one; DIMBOA, 2, 4-dihydroxy-7-methoxy-1, 4-benzoxazin-3-one; DIMBOA-glc, 2-b-D glucopyr- anosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one; ES, Electro spray nega- tive mode; FeSO 4 , Iron sulphate; G6PDH, Glucose-6-phosphate dehydrogenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HBOA, 2-hydroxy-1, 4- benzoxazin-3-one; HBOA-glc, 2-b-D-glucopyranosyloxy-1,4-benzoxazin 3-one; HMBOA, 2-hydroxy-7-methoxy-1, 4-benzoxazin-3-one; HMBOA-glc, 2-b-D gluco- pyranosyloxy-7-methoxy-1, 4-benzoxazin-3-one; IPTG, Isopropyl b-D-1- thiogalactopyranoside; LC-MS/MS, Liquid chromatography-mass spectrometry; MBOA, 6-methoxy-2-benzoxazolinone; MRM, Multiple reaction monitoring; PCR, Polymerase chain reaction; qRT-PCR, Quantitative real-time polymerase chain re- action; SPF, Putative splicing factor; TRIBOA-glc, 2,4,7-trihydroxy-1,4-benzoxazin- 3-one-glucoside; TRIBOA, 2,4,7-trihydroxy-2H-1,4-benzoxazin-3(4H)-one. * Corresponding author. Forsøgsvej 1, 4200 Slagelse, Denmark. E-mail address: Per.gregersen@mbg.au.dk (P.L. Gregersen). Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem http://dx.doi.org/10.1016/j.phytochem.2017.04.020 0031-9422/© 2017 Elsevier Ltd. All rights reserved. Phytochemistry 140 (2017) 95e107